Liquid Chromatography

A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for estimation of Ornidazole in injection formulations. The separation was achieved by using column Inertsil ODS-3V (150×4.6 mm, 5µ), in mobile phase consisted of acetonitrile and pH 3.0 phosphate buffer, adjusted to pH 3.0 with the help of dilute orthophosphoric acid in the ratio of (10:90, v/v). The flow rate was 2.0 mL/min-1 and the separated Ornidazole was detected using UV detector at the wavelength of 300 nm. Column temperature 25°C and sample temperature ambient and injection volume 20µL. The retention time of Ornidazole, was noted to be 12.05 min respectively. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for estimation of Fumaric acid in Quetiapine hemi fumarate drug substance. The separation was achieved by using column Hypersil C18 (250×4.6mm, 5µm), in mobile phase consisted of acetonitrile and pH 3.0 phosphate buffer, adjusted to pH 3.0 with the help of dilute orthophosphoric acid in the gradient elution. The flow rate was 1.0 mL/min-1 and the separated Fumaric acid was detected using UV detector at the wavelength of 210 nm. Column temperature 25°C and sample temperature ambient and injection volume 20µl. The retention time of Fumaric acid, was noted to be 3.65 min respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for simultaneous estimation of Isoniazid and Pyridoxine tablet formulations. The separation was achieved by using column Hypersil BDS, (250 x 4.6 mm, 5 µ) (Make: Thermo), in mobile phase consisted of pH4.0 phosphate buffer and Acetonitrile in the ratio of 75:25 v/v. The flow rate was 1.0 mL/min, column oven temperature 30° C, the injection volume was 10 μL, and detection was performed at 267 nm using a photodiode array detector (PDA), Run time 6 minutes. The retention time of Isoniazid and Pyridoxine, was noted to be 3.5 minutes and 4.3 minutes respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

Green Chemistry covers analysis of sample containing organic compounds in different composition of matrices by using gas chromatography or liquid chromatography method with the use of less toxic solvents. It is important that the methods used for analysis of analyte in a sample have negligible harmful environmental impact. Green Chromatography mainly focus on making the methods greener during analysis at all the steps, starting from collection of sample, to its preparation and then its separation and finally determination of analyte. The present review includes the goals to achieve green chromatography using environmentally benign solvents and reagents, most recent contribution in the development of greener sample preparation since it has effect on whole analytical methodology, chromatographic separation techniques, advantages of Green Chromatography, Green micro extraction techniques (solid phase micro extraction, still bar sorptive, liquid phase micro extraction) is included with special attention. The approach in making chromatographic separations greener differs depending on the type of chromatography which includes solvent less extraction technique- aiming to eliminate or reduce the amount of organic solvents consumed without loss in chromatographic performance. In gas chromatography it is advisable not to use helium as the carrier gas because it is a non-renewable resource. GC separations using low thermal mass technology can be considered greener as energy consumption is minimized by this technology. In liquid chromatography the focus will be on the reduction of solvent consumption and replacement of toxic and environmentally hazardous organic solvents with more benign alternatives.

A new simple, accurate, rapid and precise isocratic RP-HPLC was developed and validated for the determination of Rosuvastatin and Ezetimibe in Pharmaceutical tablet dosage form by droping method. The Method employs Shimadzu LC system on Hypersil ODS column (4.6 x 250 mm, 5 µm) and flow rate of 1.5ml/min with an injection volume 20µl.  Buffer, Acetonitrile and Methanol was used as mobile phase in the composition of 40:30:30v/v. The Detection was carried out at 230nm. Linearity ranges for Rosuvastatin and Ezetimibe were 11-33µg/ml, 10-30µg/ml respectively for HPLC. Retention Time of Rosuvastatin and Ezetimibe were found to be 3.7 and 5.7 min respectively. Percent Recovery study values of Rosuvastatin and Ezetimibe were found 99.6-101.1% and 99.9-100.7% respectively. This newly developed method i.e. droping method was successfully utilized for the Quantitative estimation of Rosuvastatin and Ezetimibe in tablet dosage form. This method was validated for selectivity, accuracy, precision, and linearity, Ruggedness, Robustness and Stability Studies as per ICH guidelines.

A specific, sensitive and rapid LCMS/MS method was developed for simultaneous determination of olmesartan and hydrochlorothiazide in human plasma using olmesartan D4 and hydrochlorothiazide 13C6 as internal standards. Solid-phase extraction (SPE) method was used to extract the analytes from biological matrix. Analysis was carried out on phenomenex Luna C18 column with a flow rate of 0.600 mL/minute with 80% flow splitting. Detection was carried out on a triple quadrupole linear mass spectrometer, equipped with turbo ion spray source. The method was validated over the concentration range of 32.32 ng/mL to 2676.60 ng/mL for olmesartan and 5.12ng/mL to 423.83ng/mL for hydrochlorothiazide. Olmesartan and hydrochlorothiazide were found to be stable upto 75 days in K3EDTA based Human Plasma at -20ºC. Inter and intra-batch precision of olmesartan and hydrochlorothiazide were less than 15% and the accuracy was within 85–115% in plasma.  The mean % recovery was 53.09 % for olmesartan and 59.12 % for hydrochlorothiazide in human plasma. The stability of olmesartan and hydrochlorothiazide in plasma were confirmed up to five freeze-thaw cycles at -20°C and on bench up to 24 hours and 15 minutes at ambient temperature. The method was validated satisfactorily and was suitable for the quantitation of olmesartan and hydrochlorothiazide from plasma samples in a pharmacokinetic study.

Triamterene is a potassium-sparing diuretic which is commonly used in combination with Hydrochlorothiazide, a thiazide-type diuretic in clinical management of edema and moderate hypertension. In this study, a rapid and sensitive LC–MS/MS method was developed and validated for determination of Triamterene and Hydrochlorothiazide from K3EDTA based Human Plasma. Triamterene D5 and Hydrochlorothiazide 13C6 were used as an Internal Standards for analysis of Plasma Samples.  The analytes and internal standards, were extracted by liquid–liquid extraction method using Se Quant®ZIC-HILIC, (5um,200A 150 X 4.6 mm)  column with Acetonitrile and 2 mM Ammonium formate containing 0.1% formic acid (80:20 v/v) as the mobile phase. Linearity was assessed from 3.10ng/mL to 229.72 ng/mL for Triamterene and 5.47 ng/mL to 405.27 ng/mL for Hydrochlorothiazide in plasma.  No significant matrix effects were observed by analysing the plasma samples on LC–MS/MS. The accuracy was in the range of 98.32 % to 102.86 % for both compounds. Triamterene and Hydrochlorothiazide were found to be stable up to 120 days in K3EDTA based Human Plasma at -20ºC. The stability of Triamterene and Hydrochlorothiazide in plasma was confirmed up to five freeze-thaw cycles (−20°C) and on bench up to 25 hours. The proposed bioanalytical method for the quantitation of Triamterene and Hydrochlorothiazide from K3EDTA based human plasma samples was satisfactorily validated. It can be used to include study data for quantitation of Triamterene and Hydrochlorothiazide from K3EDTA based human plasma in bioequivalence and bioavailability study.

A simple, rapid, accurate, precise and economical reverse phase high performance liquid chromatographic method was developed for simultaneous quantification of two anti-hypertensive drugs Olmesartan and Chlorthalidone. The separation of both the drugs was achieved on BDS C18 250mm x 4.6 mm, 5m using a mobile phase of 10 mM orthophosphoric acid buffer and acetonitrile (45:55v/v) at a flow rate of 1.0 mL min-1 and detection was performed at 212 nm using photodiode array (PDA) detector. The drug was subjected to various ICH prescribed stress conditions including hydrolysis (neutral, acid and alkaline), oxidation, photolysis and thermal degradation. The proposed method was validated with respect to specificity, linearity, accuracy, and precision, limit of detection (LOD), limit of quantitation (LOQ), stability and robustness as per ICH guidelines. The proposed analytical method could effectively separate the drug from its degradation products employed as stability indicating studies.

A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for estimation of Curcumin in tablet formulations. The separation was achieved by using column Hypersil BDS C18, 150x4.6 mm, 5µ (Make: Thermo), in mobile phase consisted of tetrahydrofuran and citric acid buffer in the ratio of (550:450, v/v). The flow rate was 1.0 mL.min-1 and the separated curcumin was detected using UV detector at the wavelength of 425 nm. The retention time of curcumin, was noted to be 8.05 min respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

A new rapid, precise and sensitive reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the estimation of Atazanavir and Ritonavir simultaneously in combined dosage form. The two components Atazanavir and Ritonavir were well resolved on an isocratic method, C18 column, utilizing a mobile phase composition of acetonitrile: 0.02M ammonium acetate buffer (40:60), v/v, pH 4.0) at a flow rate of 1.0 mL/min with UV detection at 205 nm. The retention time of Atazanavir and Ritonavir were 2.8 min and 5.7 min respectively. The developed method was validated for specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ) and robustness as per ICH guidelines. Linearity for Atazanavir and Ritonavir were found in the range of 18.0-42.0 µg/ml and 5.0-14.0 µg/ml, respectively. The percentage recoveries for Atazanavir and Ritonavir ranged from 98.9-101.0 % and 98.2-100.1 %, respectively. The proposed method could be used for routine analysis of Atazanavir and Ritonavir in their combined dosage forms.