A study of Nigella sativa induced growth inhibition of MCF and HepG2 cell lines: An anti-neoplastic study along with its mechanism of action.

OBJECTIVE: To evaluate the anticancer potential of seeds of Nigella sativa using MCF and HepG2 cell lines along with its mechanism of action. MATERIALS AND METHODS: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and acridine orange/ethidium bromide nuclear staining technique were selected to evaluate anticancer potential and mechanism of action of test extract. RESULTS: Aqueous extract of N.sativa at a test dose of 180 mg and 300 mg was identified to be the best as anticancer agent against MCF and HepG2 cell lines among different solvent test extract where doxorubicin and cisplatin were employed as standard references. DISCUSSION: Further study including separation and characterization of active principles in the aqueous extract shall prove beneficial.

A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability).

Palladium-catalyzed C-N and C-O bond formation of N-substituted 4-bromo-7-azaindoles with amides, amines, amino acid esters and phenols.

Simple and efficient procedures for palladium-catalyzed cross-coupling reactions of N-substituted 4-bromo-7-azaindole (1H-pyrrole[2,3-b]pyridine), with amides, amines, amino acid esters and phenols through C-N and C-O bond formation have been developed. The C-N cross-coupling reaction of amides, amines and amino acid esters takes place rapidly by using the combination of Xantphos, Cs(2)CO(3), dioxane and palladium catalyst precursors Pd(OAc)(2)/Pd(2)(dba)(3). The combination of Pd(OAc)(2), Xantphos, K(2)CO(3) and dioxane was found to be crucial for the C-O cross-coupling reaction. This is the first report on coupling of amides, amino acid esters and phenols with N-protected 4-bromo-7-azaindole derivatives.

N-heterocyclic carbene-mediated hydroacylation-Sonogashira/Heck/Suzuki coupling in a single pot: a new cascade reaction.

A dually NHC-catalyzed reaction cascade comprising an initial hydroacylation of an activated ketone and subsequent Sonogashira/Heck/Suzuki coupling in the same pot is reported. The reaction mechanism and scope of the methodology is presented.

A validated stability-indicating RP-HPLC assay method for Amsacrine and its related substances.

A validated specific stability indicating reversed-phase high-performance liquid chromatography method was developed for the quantitative determination of Amsacrine as well as its related substances determination in bulk samples, in presence of degradation products, and its process related impurities. Forced degradation studies were performed on bulk samples of Amsacrine as per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) prescribed stress conditions using acid, base, oxidative, thermal stress, and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during basic hydrolysis, slight degradation was observed in oxidative and thermal stress, and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process-related impurities and degradation products from the analyte were achieved on Inertsil ODS column using the mobile phase consists a mixture of 1.0% triethyl amine in 20 mM potassium dihydrogen orthophosphate, with pH adjusted to 6.5, with ortho phosphoric acid in water and acetonitrile using a simple linear gradient. The detection was carried out at wavelength 248 nm. The mass balance in each case was in between 99.4% to 99.9%, indicating that the developed method was stability-indicating. Validation of the developed method was carried out as per ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Amsacrine at the time of batch release and also during its stability studies.

Development of liquid chromatographic enantiomer separation methods and validation for the estimation of (R)-enantiomer in eslicarbazepine acetate.

Chiral separation method development was carried out for eslicarbazepine acetate and its (R)-enantiomer on diverse chiral stationary phases. Better chiral selectivity was observed on cellulose tris-(3,5-dichlorophenylcarbamate) immobilized column (Chiralpak IC-3). Under polar organic mode (POM), with 100% acetonitrile as mobile phase and 0.5 ml/min flow, a resolution close to three was achieved. With normal phase (NP) mobile phase consisting dichloromethane:ethanol (90:10, v/v) and 1.0 ml/min flow, a resolution close to six was achieved. Detection was done by UV at 220 and 240 nm respectively. Both the methods were found to be robust and were validated with respect to robustness, precision, linearity, limit of detection, limit of quantification and accuracy. The proposed methods are suitable for the accurate estimation of (R)-enantiomer in bulk drug samples up to 0.1% when a 1mg/ml analyte test solution is chromatographed.

Achiral bis-imine in combination with CoCl2: A remarkable effect on enantioselectivity of lipase-mediated acetylation of racemic secondary alcohol.

A bis-imine (prepared via a new FeCl3-based method) in combination with CoCl2 facilitated lipase-mediated acetylation of the (R)-isomer of a racemic benzylic secondary alcohol with 91% ee(s). The methodology was used for the preparation of the known drug rivastigmine.

Degradation studies of pentoxifylline: Isolation and characterization of a novel gem-dihydroperoxide derivative as major oxidative degradation product.

Pentoxifylline was subjected to various stress conditions and degradation profile was studied with conventional LCMS. Interestingly, under oxidative stress conditions the drug substance underwent distinct transformation to give rise to a single major degradation product. The structure of this product was elucidated using 1D, 2D NMR spectroscopy, high resolution mass spectrometry (Q-TOF LC/MS) and found to be a novel gem-dihydroperoxide, namely 1-(5,5-Bis-hydroperoxy-hexyl)-3,7-dimethyl-3,7-dihydro-purine-2,6-dione. An efficient stability indicating liquid chromatographic separation method was developed for pentoxifylline and its three degradation products (including two from base hydrolysis) using 1.8 microm, C18 reverse phase column and UHPLC. Baseline separation was achieved with a run time of 4 min. The analytical assay method was validated with respect to system suitability, specificity, linearity, range, precision, accuracy and robustness.

Studies on some physicochemical properties of leucaena leucocephala bark gum.

Gum exudates from Leucaena Leucocephala (Family: Fabaceae) plants grown all over India were investigated for its physicochemical properties such as pH, swelling capacity and viscosities at different temperatures using standard methods. Leucaena Leucocephala bark gum appeared to be colorless to reddish brown translucent tears. 5 % w/v mucilage has pH of 7.5 at 28°C. The gum is slightly soluble in water and practically insoluble in ethanol, acetone and chloroform. It swells to about 5 times its original weight in water. A 5 %w/ v mucilage concentration gave a viscosity value which was unaffected at temperature ranges (28-40°C). At concentrations of 2 and 5 %w/v, the gum exhibited pseudo plastic flow pattern while at 10 %w/v concentration the flow behaviour was thixotropic. The results indicate that the swelling ability of Leucaena Leucocephala (LL) bark gum may provide potentials for its use as a disintegrant in tablet formulation, as a hydro gel in modified release dosage forms and the rheological flow properties may also provide potentials for its use as suspending and emulsifying agents owing to its pseudo plastic and thixotropic flow patterns.

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant.

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz.

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.

Development and validation of a reverse-phase liquid chromatographic method for assay and related substances of abacavir sulfate.

A simple isocratic liquid chromatographic method was developed for the determination of abacavir from its related substances and assay for the first time. This method involves the usage of C18 (Intertsil octadecyl silane-3V, 150 mm x 4.6 mm, 5 microm) column. The method was validated over the range of 0.002-0.1 mg/mL for chloro impurity, 0.005-0.1 mg/mL for amino impurity and pyrimidine impurity, and 0.005-0.2 mg/mL for abacavir. The mobile phase consists of a mixture of 10 mM ammonium acetate buffer and ACN in the ratio of 90:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 214 nm. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. The developed method was validated for linearity, range, precision, accuracy, and specificity. This method can be conveniently used in a quality control laboratory for routine analysis of both related substances and assay.

Development and validation of a specific stability indicating high performance liquid chromatographic method for rizatriptan benzoate.

A gradient, reversed-phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of rizatriptan benzoate, used to treat relieves migraine headache symptoms. The developed method can be also employed for the related substance determination in bulk samples. Forced degradation studies were performed on bulk sample of rizatriptan benzoate using acid (0.5 N hydrochloric acid), base (0.1 N sodium hydroxide), oxidation (3.0% hydrogen peroxide), water hydrolysis, heat (60 degrees C) and photolytic degradation. Mild degradation of the drug substance was observed in base hydrolysis and considerable degradation observed during oxidative stress. The chromatographic method was fine tuned using the samples generated from forced degradation studies. Good resolution between the peaks corresponds to degradation products and the analyte was achieved on Agilent Zorbax SB-CN (250 mm x 4.6 mm, 5 microm) column. The mobile phase consists of a mixture of aqueous potassium di hydrogen ortho phosphate (pH 3.4), acetonitrile and methanol. The stress sample solutions were assayed against the qualified reference standard of rizatriptan benzoate and the mass balance in each case was close to 99.7% indicating that the developed method is stability indicating. Validation of the developed method was carried out as per ICH requirements.

A stability-indicating HPLC assay method for docetaxel.

A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for docetaxel in the presence of degradation products generated from forced decomposition studies. A gradient HPLC method was developed to separate the drug from the degradation products, using a Hichrom RPB HPLC column. Mixture of water and acetonitrile was used as mobile phase. The flow rate was 1.0 ml/min and the detection was done at 230 nm. Using the above method one can carry out the quantitative estimation of impurity namely DCT-1 and docetaxel. The developed gradient LC method was subsequently validated.

A validated stability indicating ion-pair RP-LC method for zoledronic acid.

The present paper describes the development of a stability indicating high performance liquid chromatographic (HPLC) assay method for zoledronic acid in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of zoledronic acid was observed under oxidative stress at higher temperature. The drug was found to be stable in other stress conditions attempted. Successful separation of the drug from the degradation products formed under stress conditions was achieved on a C18 column using a mixture of phosphate buffer that contains 7 mM tetra butyl ammonium hydrogen sulphate, an ion-pairing agent and methanol (95:5) as mobile phase. The developed HPLC method was validated with respect to response function, precision, accuracy, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of zoledronic acid can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of zoledronic acid.

A validated chiral liquid chromatographic method for the enantiomeric separation of Rivastigmine hydrogen tartarate, a cholinesterase inhibitor.

A new and accurate chiral liquid chromatographic method was developed for the enantiomeric resolution of Rivastigmine hydrogen tartarate, (-)S-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methylphenyl-carbamate hydrogen tartarate, a cholinesterase inhibitor in bulk drugs. The enantiomers of Rivastigmine hydrogen tartarate were baseline resolved on a Chiralcel OD-H (250 mm x 4.6 mm, 5 microm) column using a mobile phase system containing hexane: isopropanol: trifluoroacetic acid (80:20:0.2, v/v/v). The resolution between the enantiomers was not less than four and interestingly distomer was eluted prior to eutomer in the developed method. The presence of trifluoroacetic acid in the mobile phase has played an important role in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The limit of detection and limit of quantification of (R)-enantiomer were found to be 500 and 1500 ng/ml, respectively for 10 microl injection volume. The percentage recovery of (R)-enantiomer was ranged from 95.2 to 104.3 in bulk drug samples of Rivastigmine hydrogen tartarate. Rivastigmine hydrogen tartarate sample solution and mobile phase were found to be stable for at least 48 h. The proposed method was found to be suitable and accurate for the quantitative determination of (R)-enantiomer in bulk drugs. Chiralcel OJ-H column can also be used as an alternative for the above purpose.

A validated chiral LC method for the enantiomeric separation of Zolmitriptan key intermediate, ZTR-5.

A new and accurate chiral liquid chromatographic method was described for the enantiomeric separation of ZTR-5 [(4S)-4-(4-aminobenzyl)-2-oxazolidinone, (S)-isomer], a key intermediate of Zolmitriptan in bulk drugs. The enantiomers of ZTR-5 were baseline resolved on a Chiralpak AD-H (250 mm x 4.6 mm, 5 microm) column using a mobile phase system containing hexane:ethanol (70:30, v/v). The resolution between the enantiomers was not less than four and interestingly distomer was eluted prior to eutomer. The limit of detection and limit of quantification of (4R)-4-(4-aminobenzyl)-2-oxazolidinone [(R)-isomer] were found to be 250 and 750 ng/ml, respectively, for 10 microl injection volume. The percentage recovery of (R)-isomer ranged from 92.0 to 105.6 in the bulk drug samples of ZTR-5. The validated method yielded good results regarding precision, linearity, accuracy and ruggedness. The proposed method was found to be suitable and accurate for the quantitative determination of (R)-isomer in bulk drug samples of ZTR-5.

A stability indicating LC method for zolmitriptan.

A gradient, reversed-phase liquid chromatographic (RP-LC) assay method was developed for the quantitative determination of zolmitriptan, used to treat severe migraine headaches. The developed method is also applicable for the related substances determination in bulk drugs. The chromatographic separation was achieved on a Waters X Terra RP18, 250 mm x 4.6 mm, 5 microm column. The gradient LC method employs solutions A and B as mobile phase. The solution A contains a mixture of phosphate buffer pH 9.85:methanol:acetonitrile (70:20:10, v/v/v) and solution B contains a mixture of phosphate buffer, pH 9.85:acetonitrile (30:70). The flow rate was 1.0 ml/min and the detection wavelength was 225 nm. In the developed HPLC method, the resolution between zolmitriptan and its potential impurities, namely Imp-1, Imp-2 and Imp-3 was found to be greater than 3. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in alkaline medium and oxidative stress conditions. Degradation product formed during base hydrolysis was found to be Imp-3. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

A validated chiral LC method for the determination of zolmitriptan and its potential impurities.

A new, accurate and reliable chiral HPLC method was developed for the determination of Zolmitriptan, (4S)-4-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl] methyl]-2-oxazolidinone an antimigraine agent and its potential impurities namely (4R)-4-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl] methyl]-2-oxazolidinone [(R)-enantiomer] and (4S)-4-(4-aminobenzyl)-2-oxazolidinone (Imp-1) in pharmaceutical formulations and in bulk drugs. HPLC separation was carried out by normal phase chromatography with a mobile phase composed of hexane:isopropanol:methanol:diethylamine in the ratio (75:10:15:0.1, v/v/v/v) pumped at a flow rate of 1.0 ml/min on a Chiralpak AD-H column. Zolmitriptan and its potential impurities were baseline resolved in the optimized method. The presence of diethylamine in the mobile phase has played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. The developed method was also found to be selective under exposed conditions UV light and 60 degrees C. The developed method was completely validated and proved to be robust. The values of the limit of detection (LOD) and limit of quantification (LOQ) of (R)-enantiomer and Imp-1 were 100, 250 ng/ml and 30, 1000 ng/ml, respectively, for 10 microl injection volume. The validated method yielded good results regarding selectivity, linearity, precision, accuracy and ruggedness. Zolmitriptan sample solution and mobile phase are found to be stable for at least 24 h. The proposed method was found to be suitable and accurate for the quantitative determination of Zolmitriptan and its impurities namely (R)-enantiomer and Imp-1 in bulk drugs and commercial formulations.