HPLC

A total of 25 different types of actinomycetes were isolated from the soils of marine sediment soil. All the isolated actinomycetes were characterized and identified based on the morphological, biochemical, cultural characteristics. Both primary and secondary screening methods were used to screen actinomycetes for antibacterial activity. The result of the screening revealed that all the isolates were against bacterial culture. But the best strain was found to be Streptomyces sp as they showed broad spectrum activity with big zone of inhibition, even though the strain Staphylococcus and Streptomyces sp showed augmented antibacterial activity against all the tested human bacterial pathogens. Comparatively, when they were treated with pathogenic microorganisms all the isolates produced to maximum and minimum zone of inhibition with its responsible broad spectrum of bioactivity. Moreover, LAM-4 and LAM-11 strains were clearly showed significant activity against both Staphylococcus aureus and S. agaricus. From the HPLC peaks which had antimicrobial activity of Streptomyces sp was identified to be at 3.296. This is similar to oxohexaene antibiotic whereas, the antimicrobial compound of Streptomyces sp3 showed a retention time of 3.233 on HPLC, this peak was similar to Cephalexin. From the results highlight the most of the isolates inhibited growth of the Gram negative bacteria tested. All the antibiotic producing actinomycetes were isolated at different temperatures from marine soil. These microorganisms might have potential to produce a number of the most important medicines constantly developed. Key words- Actinomycetes, pathogenic organisms, HPLC

A simple, specific, selective and accurate stability-indicating reversed phase high performance liquid chromatographic method was developed for simultaneous determination of Diclofenac potassium (DIC), Paracetamol (PCM) and Methocarbamol (MET). An isocratic RP-HPLC was achieved on younglin HPLC system using Varian C18 (250 Χ 4.6 mm i.d, 5 μm particle size) column with the mobile phase containing mixture of Methanol:water (80:20,v/v). The flow rate was 0.8 ml/min and the eluent was monitored at 225nm. The retention times of DIC, PCM and MET were found to be 3.51, 6.42 and 9.90 min respectively. The linearity was established for DIC, PCM and MET in the range of 10-60 µg/ml, 65-390µg/ml, 100-600µg/ml respectively. The percentage recoveries of DIC, PCM and MET were found to be in the range of 99.73%±0.109, 99.59%±0.085 and 99.50%±0.16 respectively. The LOD for DIC, PCM and MET were found to be 0.15, 2.40 and 1.82μg/ml respectively, while LOQ were 0.48, 7.29 and 5.53μg/ml respectively. All three drugs were subjected to acid, alkali, oxidation, and dry heat degradation. The degradation studies indicated DIC, PCM and MET showed degradation in acid, alkaline, H2O2, and in dry heat condition. The degradation products of DIC, PCM and MET were resolved well from the pure drug with significant differences in their retention time values. This method was also successfully employed for simultaneous quantitative analysis of DIC, PCM and MET in bulk drugs and formulations. The developed method is stability indicating and separate degradants and can be used to determine the stability of samples.

A simple, specific, accurate and stability-indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of thiocolchicoside  and lornoxicam, using a RP-18 column and a mobile phase composed of 10mM ammonium acetate : methanol(50:50), pH7 adjusted with 1%triethyl amine. The retention time of thiocolchicoside and lornoxicam were found to be 4.6 and 10.2 min, respectively. Linearity was established for both thiocolchicoside and lornoxicam in the range of 1-10 µg/ml. The percentage recoveries of thiocolchicoside and lornoxicam were found to be 100.45±0.4489 and 100.70±0.5111, respectively. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, and photolytic degradation. The degradation studies indicated thiocolchicoside to be susceptible to acid, alkaline and neutral hydrolysis while lornoxicam showed degradation under acid and alkali. The degradation products of thiocolchicoside and lornoxicam 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 thiocolchicoside and lornoxicam in bulk drugs and formulations.

An accurate, simple, precise and sensitive HPLC method with UV detection was developed and validated to separate and detect ibuprofen in human plasma using Nimesulide as an internal standard. Ibuprofen and Nimesulide were extracted from human plasma using acetonitrile protein precipitation and HPLC analysis was performed using Waters 515 Series pumps combined with a Waters PDA 2998 series photo diode array detector (DAD). The column used was Agilent C18 column (150mm×4.6mm, particle size 5-micron Agilent, USA). Analysis was isocratic at 1.5 ml/min flow rate with ACN: Buffer (0.025M Potassium dihydrogen ortho phosphate) pH 4.5 (55:45, v/v) as mobile phase. The mobile phase was premixed, filtered through a 0.2 µm nylon membrane filter to remove any particulate matter and degassed by sonication before use. The elution was detected at 230 nm. Each solution was injected in triplicate, and the relative standard deviation (R.S.D.) was measured. The retention times of Ibuprofen 2.24 min and for I.S. 1.72 min respectively. The method was validated over the range of 0.5-8.0 μ/ml. The limit of detection was 0.06μg/ml and the limit of quantification was 0.193μg/ml for ibuprofen. Inter-day as well intra-day replicates of Ibuprofen, gave % R.S.D. below 2.07 and 2.001 respectively  The absolute recovery of ibuprofen was greater than 90% were achieved. This method of analysis for Ibuprofen determination using RP-HPLC was applied for determination of Ibuprofen in plasma.

A rapid and sensitive High Performance liquid chromatography (HPLC) method has been developed and validated. The analyte was extracted from human plasma by simple precipitation technique. Nifedipine was used as the internal standard. A Princeton C18 column provided chromatographic separation of the analyte. A simple, selective, rapid, precise and economical reverse phase High Pressure Liquid Chromatographic method has been developed for the estimation of Lercanidipine in human Plasma. The method was carried out on a Princeton C18 (250 mm x 4.6 mm i.d., 5µ) column with a mobile phase consisting of acetonitrile: Water (adjusted to pH 3.5 using orthophosphoric acid) (55:45 v/v) at a flow rate of 1.0 ml/min. Detection was carried out at 235 nm. The retention time of Lercanidipine Nifedipine, was 5.31, 10.00 min, respectively. The proposed method has been validated with linear range of 5.0-250.0 ng/ml for Lercanidipine. The precision and accuracy values are within 10%. The overall recovery of Lercanidipine was 96.4 %. The developed and validated method was applicable for the pharmacokinetics studies.

  A simple, fast, and precise reverse phase, gradient HPLC method was developed for the separation and quantification of metformin hydrochloride and its related compounds Cyanoguanidine impurity, Melamine impurity, 1- methylbiguanidine impurity, Monoguanylmelamine impurity, N,N Dimethylmelamine impurity in tablet formulations. Liquid chromatography with using Partisil SCX, 250 X 4.6 mm, 10µm and mobile phase is 17 gms of ammonium Dihydrogen phosphate in 1000 ml water and adjust the pH to 3.0 with phosphoric acid and degassed under sonication. The flow rate was 1.0 ml/min and the effluent was monitored at 218 nm. This new method was validated in accordance with USP requirements for new methods for assay determination, which include accuracy, precision, linearity, range and robustness. The current method demonstrates good linearity over the range of 0.01µg/mL to 10µg/mL for Impurity A and metformin HCl. Remaining all impurities with concentration from 0.02 µg/mL to 15 µg/mL for six levels. The accuracy is carried out with concentrations ranging from 50% to 200% of Target concentration the Mean % recovery for each impurity at each level should be between 85.0 % and 115.0.The precision of this method reflected by relative standard deviation of replicates all metformin Related Substances is NMT 10%. Validation of the same method was also performed according to USP requirements for quantitative determination of impurities which include robustness and limit of quantification (LOQ) and Limit of detection LOD. No significant variation in RRT of Metformin and its substances at flow rate (0.8 to 1.2mL/min.), at pH (2.8 to 3.2), column temperature (23°C to 27°C), hence the method is robust. Key words: Metformin Hydrochloride, Metformin related substances, HPLC, method validation.

  In the present work the approach of forced degradation study was successfully applied for the development of stability-indicating assay method for determination of Thiocolchicoside in the presence of its degradation products.  The RP-HPLC separation was carried out on Shimadzu® -HPLC 1100 series using a Phenomenex ODS 5µ C18 column (250×4.6mm) with mobile phase comprising of Acetonitrile: Phosphate Buffer (70:30) pH 3.5 v/v at flow rate of 1.0mL/min and UV detection at 260.0 nm. In stress testing a drug substance or the drug product is exposed to an environment vigorous than the normal i.e. at high temperature, high humidity over the period of time called accelerated stability conditions. The drug was subjected to Solid state analysis which includes Humidity studies (40°C/75% RH), photochemical studies (UV light and sunlight exposure) and Thermal studies to apply stress conditions. The method was validated as per ICH guidelines for accuracy, precision, linearity and range, ruggedness and robustness. The linearity of the proposed method was investigated in the range of 80-120% of label claim; the correlation coefficient for Thiocolchicoside was found to be 0.999. The proposed method was found to be simple, specific, linear and rugged and can be used for routine quality control.