A validated chiral LC method for the enantioselective analysis of Levetiracetam and its enantiomer R-alpha-ethyl-2-oxo-pyrrolidine acetamide on amylose-based stationary phase.

A new, simple chiral HPLC method was developed for the enantiomeric separation of Levetiracetam, [(S)-alpha-ethyl-2-oxo-pyrrolidine acetamide], an antiepileptic drug in pharmaceutical formulations and in bulk materials. Enantiomeric separation was achieved on a chiralpak AD-H column using a mobile phase consisting of hexane and isopropanol in the ratio (90:10, v/v) at a flow rate of 1.0 ml/min. The resolution between the enantiomers was found to be not less than 7 in the optimized method. Interestingly, unwanted enantiomer, namely R-alpha-ethyl-2-oxo-pyrrolidine acetamide ((R)-enantiomer), was eluted prior to its mirror image in the developed method. The developed method was found to be selective in the presence of related impurities of Levetiracetam, namely N-(1-carbamoyl-propyl)-4-chloro-butyramide (Imp-1) and 1-ethyl-2-oxo-1-pyrrolidine acetic acid (Imp-2), and also under exposed conditions of UV light and 60 degrees C. The limit of detection (LOD) and limit of quantification (LOQ) of (R)-enantiomer were found to be 900 and 2250 ng/ml, respectively, for 10 microl injection volume. The method precision for (R)-enantiomer at limit of quantification level was within 8% R.S.D. Calibration curve for (R)-enantiomer was linear over the studied ranges (2250-9000 ng) with correlation coefficient greater than 0998. The active pharmaceutical ingredient was extracted from its finished dosage form (tablet) using isopropanol. The percentage recoveries of (R)-enantiomer were ranged from 94.2 to 102.6 and from 93.5 to 104.1 in spiked bulk and formulation samples of Levetiracetam, respectively. Levetiracetam sample solution and mobile phase are found to be stable for at least 48 h. The developed method was found to be rugged and robust. The proposed method was found to be suitable and accurate for the quantitative determination of (R)-enantiomer in bulk drugs and commercial formulations. Chiralcel OD-H column can also be used as an alternative column for the above purpose.

Structural studies on the impurities of troglitazone.

The impurity profile study of troglitazone has been carried out primarily by (liquid chromatography-mass spectrometry) LC-MS. Four process-related impurities have been detected by LC-MS and were confirmed by co-injection with authentic samples. Apart from the process-related impurities, two polar by-products were characterized by mass spectral data and comparison with reference samples, while one non-polar by-product and one degradation product have been isolated by means of preparative HPLC and characterized by 2D NMR and mass spectral study. Single-crystal X-ray diffraction studies have been carried out on the degradation product. The formation and characterization of these by-products and degradation product are discussed.

Isolation and characterization of process-related impurities in linezolid.

Two unknown impurities in linezolid bulk drug at levels below 0.1% (ranging from 0.05 to 0.1%) were detected by a simple isocratic reverse phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of linezolid using reverse phase preparative HPLC. Based on the spectroscopic data (IR, NMR and MS) the structures of the impurities were characterized as (S)-N-[[-(3-(3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] acetate(I) and (S)-N-[[-(3-(3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] chloride(II). The synthesis from an unambiguous route and the formation of impurities was discussed.

Determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by HPLC and MEKC methods.

High Performance Liquid Chromatographic (HPLC) and Micellar Electrokinetic Chromatographic (MEKC) methods have been developed for the determination of pioglitazone, a new englycemic antidiabetic agent. Pioglitazone and its unsaturated impurity were separated by MEKC in less than 7 min using a 43 cm x 50 microm i.d. uncoated fused-silica capillary with extended light path for better sensitivity (25 kV at 30 degrees C) and a background electrolyte (BGE) consisting of 20% acetonitrile (v/v) in 20 mM sodium borate buffer pH 9.3 containing 50 mM sodium dodecyl sulphate (SDS). The influence of various parameters on the separation such as pH of the buffer, SDS concentration, buffer concentration, organic modifiers, temperature and voltage were investigated. The MEKC method was compared with HPLC method using a 5 microm symmetry C18 column (250 x 4.6 mm i.d.) eluted with a mobile phase consisting of a mixture of 50% (v/v) acetonitrile and 10 mM potassium dihydrogen phosphate buffer, adjusting the pH to 6.0 with 0.1 M KOH. The HPLC method is capable of detecting all process related compounds, which may be present at trace levels in finished products. Both methods were fully validated and a comparison was made. The results confirm that the methods are highly suitable for its intended purpose.

Simultaneous determination of fexofenadine and its related compounds by HPLC.

A simple reversed phase liquid chromatographic (RPLC) method has been developed and subsequently validated for the determination of fexofenadine hydrochloride and its related compounds A and B. The method utilizes a C8 column for the separation and determination of meta-isomer (related compound B). The separation was achieved using an Eclipse XDB C8, 5 microm, 4.6 x 150 mm column and a mobile phase comprising 1% triethylamine phosphate (pH 3.7), acetonitrile and methanol in the ratio 60:20:20 (v/v/v). 5-Methyl 2-nitrophenol has been used as internal standard for the purpose of quantitation of fexofenadine. The described method was linear over a range of 0.7-18.7 microg/ml for related compounds A and B and 60-750 microg/ml for assay of fexofenadine. The relative standard deviation (n=3) was 0.5% for the drug and 3.4% for related compounds. The intermediate precision was 0.79% (n=9) for assay and 5.16% (n=9) for related impurities. The mean recovery of both the related compounds were in the range of 94-103%. Limits of detection (LOD) and quantification (LOQ) for the related compounds A and B were 0.18, 0.12 and 0.56, 0.48 microg/ml, respectively. The precision of the method was checked by F-test using a reported method as reference and the calculated value (1.35) was found to be less than the table value at 95% confidence levels. The obtained results confirm that the method is highly suitable for its intended purpose.

Isolation and characterisation of process-related impurities in rofecoxib.

Two unknown impurities in rofecoxib bulk drug at levels below 0.1% were detected by a simple isocratic reverse phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of rofecoxib using reverse phase preparative HPLC. (1)H, (13)C and Mass spectroscopic investigations revealed the structures of the impurities as 4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one (I) and 4-[4-(methylsulphonyl)phenyl]-3-phenyl-2,5-furandione (II), respectively. These structures were further confirmed by prepared synthetic standards of the impurities. The tentative mechanism for the formation of these impurities was discussed.

LC determination of rofecoxib in bulk and pharmaceutical formulations.

An isocratic reversed phase-liquid chromatographic (RP-LC) method has been developed for the determination and purity evaluation of rofecoxib in bulk and pharmaceutical dosage forms using photodiode array detection set at 225 nm. The method is simple, rapid and selective. The method is capable of detecting all process intermediates and other related compounds, which may be present at trace levels in finished products. Hence the method is very useful for process monitoring during the production of rofecoxib. Chlorophenyl methyl sulphone has been used as internal standard for the quantitative determination of rofecoxib. The method is linear in the range of 125-500 microg. The precision for inter- and intra-day assay variation of rofecoxib is below 1.6% relative standard deviation (R.S.D.). The accuracy determined as relative mean error (R.M.E.) for the intra-day assay is within +/-2.0%. The drug was extracted from tablets (Vioxx) using acetonitrile. The percentage recoveries from dosage forms were ranged from 98.2 to 102.6.

LC determination and purity evaluation of nefazodone HCl in bulk drug and pharmaceutical formulations.

A simple, selective and reproducible reversed-phase liquid chromatography (LC) method has been developed for the quantitative determination of nefazodone hydrochloride (I) in the presence of its related impurities, namely 5-ethyl-4-(2-phenoxyethyl)-2H-1,2,4-triazol-3-(4H)one (II), 1-(3-chlorophenyl)-4-(3-chloropropyl) piperazine hydrochloride (III) and 1,1(1)-trimethylene-bis[4-(3-chlorophenyl) piperazine] hydrochloride (IV). The separation was achieved using an Inertsil ODS-3V (250 x 4.6 mm(2)) column and a mobile phase comprising 0.05 M KH(2)PO(4) (pH 3.0), acetonitrile and methanol in the ratio 50:40:10 (v/v/v). The method has been completely validated and proven to be rugged. The limit of detection and limit of quantification for impurities II, III and IV were found to be 50, 79 and 91 ng/ml, and 152, 240 and 280 ng/ml, respectively. The intra- and inter-day assay precision of the method was within 1.2% relative standard deviations. The developed method was applied to the pharmaceutical dosage form (Tablet, Serzone-R) and the percentage recoveries ranged from 99.1 to 100.7. The percentage recovery of impurities ranged from 96.2 to 108.9. The stability studies were performed for nefazodone solution placed on laboratory bench and in the refrigerator for 60 days. The method was proved to be stability indicating in solution.

LC separation of ortho and meta isomers of celecoxib in bulk and formulations using a chiral column.

A normal phase, isocratic LC method was developed for the separation of positional isomers of celecoxib (I) using a chiral column, Chiralpak-AD. The method is useful for the quantification of ortho (II) and meta (III) forms in bulk drugs and formulation samples of celecoxib. The method has been completely validated and proven to be rugged. The limit of detection (LOD) and limit of quantitation (LOQ) of ortho and meta forms were found to be 38 ng and 116 ng respectively. The active pharmaceutical ingredient was extracted from its finished dosage form (capsule) using ethanol. The percentage recoveries of ortho isomer was found to be 99.8--102.7 and 97.8--103.2 and the percentage recoveries of meta isomer was found to be 99.3--102.6 and 99.7--104 in spiked bulk and formulation samples of celecoxib respectively.

Sparfloxacin, an antibacterial drug.

The title compound, sparfloxacin or cis-5-amino-1-cyclopropyl-7-(3,5-dimethylpiperazin-1-yl)-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid trihydrate, C(19)H(22)F(2)N(4)O(3).3H(2)O, is an antibacterial drug. The molecule, which crystallizes as a trihydrate, is in the zwitterionic form in the solid state. Hydrogen bonds stabilize the molecules in the lattice.