HPLC

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of acetaminophen level in human plasma using caffeine as an internal standard (IS) was developed and validated. 0.5 ml plasma samples containing acetaminophen were mixed with 50 µg of the IS. After adding 30 µl of 50% perchloric acid, the mixture was vortexed for one minute and then centrifuged for 5 minutes at 13200 rpm. The clear supernatant was transferred into an auto-sampler vial and 100 µl was injected into the HPLC system with a run time of 7.0 min. The compounds of interest were efficiently separated on Symmetry C18 (4.6 x 150 mm, 5-µm) column, and were detected with a photodiode array detector set at 245 nm. The mobile phase consisted of water, methanol, and acetonitrile (80:10:10, v:v:v) and was delivered at a flow rate of 0.9 ml/min. No interference in blank plasma or by commonly used drugs was observed; and the detection limit of acetaminophen was 0.05 µg/ml. The relationship between acetaminophen concentration in plasma and peak area ratio of acetaminophen /IS was linear (r2 ≥ 0.9991) in the range of 0.1– 40 µg/ml. Intra- and inter-day coefficient of variations (CV) and biases were ≤11.6%  and  ≤10.8%, and ≤≤14.0 and ≤12.8, respectively. Extraction recovery of acetaminophen and the IS from the plasma samples was ≥99% and 86%, respectively. Using the method, acetaminophen was found to be stable under conditions generally encountered in the clinical laboratory (≥99% and 91% in processed and unprocessed samples, respectively). Further, the method was successfully used to measure acetaminophen level in plasma samples from a healthy volunteer.

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of meloxicam in human plasma was developed and validated. Using piroxicam as an internal standard (IS), separation was achieved on Symmetry shield RP-18 column. 0.5 ml plasma samples were prepared by protein precipitation using trifluoroacetic acid and acetonitrile. The mobile phase consisted of 0.025 M dibasic potassium phosphate (pH=6.0, adjusted with phosphoric acid), methanol, and acetonitrile (73:5:22, v:v:v) and was delivered at a flow rate of 1.5 ml/min. Eluents were measured using photodiode array detector set at 355 nm. Under these conditions, no interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of meloxicam in plasma and peak height ratio of meloxicam to the IS was linear over the range of 0.05-2.0 μg/ml. Intra-day and inter-day coefficient of variations (CV) and biases were ≤ 6.0% and ≤ 8.6%, and ±5.9% and ±5.3%, respectively. Extraction recovery of meloxicam and the IS from plasma was ≥80% and 98%, respectively. The method was applied to assess the stability of meloxicam under various clinical laboratory conditions. In processed samples, meloxicam was stable for at least 24 hours at room temperature (≥ 82%) and 48 hours at -20C (≥ 95%). In unprocessed sample it was stable for at least 24 hours at RT (≥ 82%), 16 weeks at -20ºC (≥ 87%), and after three freeze-thaw cycles (≥ 90%). The method is suitable for clinical and bioavailability investigation involving meloxicam concentration in the therapeutic range.

A simple, specific, accurate and stability-indicating reversed phase High Performance Liquid Chromatographic method was developed for the simultaneous determination of Clindamycin Phosphate and Benzoyl Peroxide, using a C18 column and a mobile phase composed of 20 mM Ammonium acetate buffer pH 4.0: Methanol (45: 55 %v/v) as mobile phase at flow rate of 1.2 ml/min with detection wavelength of 210nm. Retention times in RP-HPLC method were found to be 4.49 min, 8.78 min for Clindamycin Phosphate and Benzoyl Peroxide, respectively. Linearity of Clindamycin Phosphate and Benzoyl Peroxide were found in the range of 10.0-30.0 µg/ml and 25.0-75.1 µg/ml. The % recovery of Clindamycin Phosphate was found to be 98.45- 101.0 and 99.8- 99.38 for Benzoyl Peroxide. Both the drugs were subjected to acid, alkali, oxidation, thermal and sunlight degradation. The degradation products of Clindamycin Phosphate and Benzoyl Peroxide were well resolved from the pure drugs with significant differences in the retention time values. This method can be successfully employed for simultaneous quantitative analysis of Clindamycin Phosphate and Benzoyl Peroxide in gel formulation. The literature survey reveals that currently there is no stability indicating method has been reported for combination of Clindamycin Phosphate and Benzoyl Peroxide till date, Which can be applicable for routine analysis of combined formulation of drugs in quality control laboratories.

A simple, rapid and specific High performance liquid chromatography (HPLC) method has been developed for simultaneous analysis of Curcumin and β-Boswellic acid in a prepared polyherbal gel formulation containing turmeric and boswellia extracts. High performance liquid chromatography analysis was performed on a C18 column using 90:10 (v/v) mixture of acetonitrile and water as isocratic mobile phase at a flow rate of 1 ml/min. Ultra violet detection was set at 425 nm for Curcumin and 242 for β-Boswellic acid. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International Conference on Harmonization guidelines. Validation data reveals that the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2>0.9993) were obtained for calibration plots in the range tested. Limit of detection for Curcumin was 0.16 µg/ml and limit of quantification was 0.50 µg/ml while for β-Boswellic acid limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.3 and 9.9 µg/ml respectively. Recovery was found to be between 98.75 to 99.01 % for Curcumin and from 98.72 to 100.01 % for β-Boswellic acid. The established HPLC method is appropriate and the two selected markers are well resolved, enabling efficient quantitative analysis of Curcumin and β-Boswellic acid. The method was successfully used for quantitative analysis of these two marker constituents in an in-house prepared polyherbal gel formulation. Key word: HPLC, Simultaneous analysis, Polyherbal gel formulation, Curcumin, β-Boswellic acid

A simple, precise, accurate, simultaneous and stability-indicating HPLC method developed with an effective resolution of active pharmaceutical ingredients. The present method effectively separates all the related substances of Diphenhydramine hydrochloride and acetaminophen along with impurities. Chromatographic separation has been obtained on Inertsil C18 (250 X 4.6mm, 5µ) column using a gradient elution with a mixture of phosphate buffer pH3.0 and acetonitrile. At 220 nm compounds will be eluted and monitored. Diphenhydramine hydrochloride and acetaminophen were subjected to the stress conditions of acid, base, peroxide, thermal, photolytic, humidity and water degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating ability of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines and validation acceptance criteria were met in all cases. The current method has proven good linearity and accuracy over the range of all known impurities from LOQ to 150% of the target concentration. The degree of reproducibility, as results obtained by deliberate changes in the method parameter and variety of condition has proven that the method is robust and rugged.

This study investigates the stability of paracetamol pediatric oral suspension (120mg/5ml) in simulated in-home storage conditions at temperature ranging from (2-8C0) representing refrigerator condition. Samples from suspension were assayed and tested for related substance (degradants) using B.P pharmacopeia HPLC method. The study was performed in day zero, seven, fourteen, thirty and forty five.  The instrument employs column ® C8, 100 x 4.6 mm, 3.5 μm particle size. The mobile phase consisted of methanol, tetrabutylammonium hydroxide (40 %) and sodium orthophosphate buffer. The results showed that the drug assay content remains within the limits up to day fourteen. Furthermore, the half live was found to be 36.8 days.

Accelerated stability studies of Emblica officinalis, Terminalia belerica and Terminalia chebula have been carried out as per ICH guidelines and its effect on total polyphenol content as determined by Folin-Ciocalteu method and Gallic acid content as determined by HPLC (High Performance Liquid Chromatography) was studied. The samples were kept in stability chamber at 40°C and 75% relative humidity for 3 months for accelerated stability studies. Samples were taken out at periodic intervals and extracted to determine total polyphenol content by spectrophotometric method and gallic acid content by HPLC. The HPLC method was also validated to demonstrate its selectivity, linearity, precision, and accuracy. The results indicate an increase in total polyphenolic content as well gallic acid content under accelerated stability conditions which is indicative of hydrolysis of gallotannic acids present in crude drugs to liberate free gallic acid, thereby increasing the total polyphenolic content.

The chromatographic conditions were successfully developed for the separation of by artemether and lumifantrine using  symmetry C18 column (4.6×150mm) 5µ,  A wavelength of 290 nm was selected and the mobile phase consists of triehtylamine  buffer (PH 6 adjusted with ortho phosphoric acid) and methanol in 20:80 % v/v ratios at a flow rate of 0.8 ml/min were found to be optimum conditions for analysis. The peaks were well resolved with symmetry C18 column. System suitability studies were also carried out which includes theoretical plates, resolution and tailing factors etc. The accuracy studies were shown as % recovery for Artemether and Lumefantrine at 50 %, 100 % and 150 %. The limits of % recovered should be in the range of 98-102 %. The results obtained for Artemether and Lumefantrine were found to be within the limits. Hence the method was found to be accurate. The accuracy studies showing % recovery of Artemether were found to be 99.9 %, 99 % and 99.9. % respectively and the % recovery of Lumefantrine were found to be 99.9%, 99.9 % and 98.9 % respectively. In the System precision study, the % RSD was found to be less than 2 %. For Artemether 1.7 and Lumefantrine 1.2 which indicates that the system has good reproducibility. For ID precision studies six replicate injections of Artemether and Lumefantrine were performed. The % RSD was determined for peak areas of Artemether and Lumefantrine. The acceptance limit should not be more than 2 % and the results were found to be within the acceptance limits. Using the optimized chromatographic conditions, chromatograms of mixed standard solutions which contained Artemether and Lumefantrine were recorded. Retention times of Artemether and Lumefantrine were found to be 2.9 min and 4.6 min respectively. Calibration curves were obtained by using peak area vs. concentration. Artemether and Lumefantrine show linearity in the range of 0.1 – 0.5 µg/ml and 0.6 – 3.0 µ/ml. Calibration curve was plotted and correlation co-efficient for both the drugs Artemether and Lumefantrine were found to be 0.999 and 0.999 respectively

Butea monosperma and Butea superba belonging to the family, Fabaceae are most exploited medicinal plants by different tribal groups of Jharkhand. They are commonly found in the hills and jungles of Jharkhand and are used against arthritis, osteoarthritis, diarrhoea, dysentery, snakebite, male sexual debilities, sunstroke, leucorrhoea, anthelmintic and filariasis. Ethanolic exacts of the barks and flowers of both the plants did not exhibit significant antibacterial and antifungal activities. Phytochemical analyses revealed a total of 14 bioactive compounds from the barks and flowers of B. monosperma and B. superba.  Successful management of several diseases among the ethnic groups of Jharkhand, is indicative of presence of curative drugs without toxicity and side effects, and it could further the isolation and purification of active compounds contained in them.

Two novel methods having requisite precision, accuracy, specificity and robustness were developed and validated for quantitative determination of Apixaban in pharmaceutical dosage forms. The first method was based on isocratic reverse phase liquid chromatography using Sunfire C18, 150mm×4.6mm, 5µ and mobile phase consists of Buffer: acetonitrile (60:40) at a flow rate 1ml/min and detection was achieved photodiodide array detector set at 280 nm. The response was linear range of 5-50 µg/ml (R2 =0.9998). The second spectrophotometric method involves detection at 280 nm. The calibration curve range between 5-50 µg/ml (R2 =0.9999). Validation of method was carried out fulfilling ICH guidelines. Both the methods were applied without any interference from excipients, for determination of drug in coated tablets. It is suggested that the proposed HPLC and UV spectrophotometric methods could be used routine quality control and dosage form assay of Apixaban.