An improved and validated LC method for resolution of bicalutamide enantiomers using amylose tris-(3,5-dimethylphenylcarbamate) as a chiral stationary phase

An improved HPLC method for determination of enantiomeric purity of bicalutamide in drugs and pharmaceuticals was developed and validated. Baseline separation with resolution >/=6.0 was achieved within 10 min on Chiralpak AD-H (250 mm x 4.6 mm; particle size 5 microm) column using n-hexane:2-propanol (65:35 v/v) as mobile phase at a flow rate of 1.0 ml/min at 25 degrees C. The detection was made at 270 nm using UV detector while a polarimetric detector connected in series was used for identification of enantiomers. The effects of 2-propanol, ethanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was validated in terms of accuracy, precision and linearity in the range of 10-250 microg/ml and the r(2) was >0.9999. The recoveries were 99.68-100.25% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) of enantiomers were (2.4, 3.0 and 7.6, 9.3) x 10(-8)g/ml for (S)-(+)-BCT and (R)-(-)-BCT enantiomers, respectively. The method was found to be suitable for rapid determination of enantiomeric purity of bicalutamide in bulk drugs and pharmaceutical formulations.     

Enantioselective determination of a gastroprokinetic drug using amylose tris-(3,5-dimethylphenylcarbamate) as a stationary phase by HPLC

An enantioselective high-performance liquid chromatographic method for determination of enantiomers of mosapride citrate in bulk drugs and pharmaceuticals using UV-vis and polarimetric detectors in series has been developed. Baseline separation with resolution >2.0 was achieved on a column containing amylose tris-(3,5-dimethylphenylcarbamate) as stationary phase using a mobile phase consisting of n-hexane:ethanol:triethylamine (80:20:0.3, v/v/v) at 40 degrees C. The detection was carried out at UV-276 nm and enantiomers were identified by polarimetric detector. The effect of ethanol, 2-propanol, TEA, temperature and mobile phase flow rate on separation of MSP enantiomers was studied and the method was validated with respect to accuracy, precision, linearity and limits of detection and quantification. The linearity of the method was studied between 6.25 and 50 microg/ml and r2 was >0.9997. The recoveries were in the range 99.63-100.22%, the method was suitable not only for process development of mosapride citrate but also for quality assurance of the individual enantiomers in bulk drugs and pharmaceuticals.     

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.     

An enantiospecific HPLC method for the determination of (S)-enantiomer impurities in (R)-tolterodine tartarate

A high-performance liquid chromatographic method was developed for the separation of the enantiomers of tolterodine tartarate. The proposed method was applied to the determination of (S)-isomer in (R)-tolterodine tartarate, and satisfactory results were obtained. The enantiomers of tolterodine tartarate were separated on a Chiralpak AD-H (250 mm x 4.6 mm) column containing amylose tris-(3,5-dimethylphenyl-carbamate) at room temperature. The mobile phase consisted of n-hexane and isopropyl alcohol in the ratio of 85:15 (v/v) with 0.075% triethylamine (TEA) and 0.05% trifluoroacetic acid (TFA) as the additive. The flow rate was kept at 0.5 ml/min, and UV detection wavelength was set at 283 nm. The calibration curves of (S)-enantiomer in the concentration range from 0.05 microg/ml to 1 microg/ml range were linear. The relative standard deviations of within-day and between-day were less than 2% (n = 3). The limit of detection (LOD) was 0.75 ng (S/N = 3) and the limit of quantification (LOQ) was 0.05 microg/ml (RSD < 4.1%, n = 3). The determination recoveries of the (S)-enantiomer were in the range of 98.2-104.8%. The results demonstrated that the developed HPLC method was a reliable, simple technique and was applicable to the purity determination of (R)- tolterodine tartarate.     

Enantiospecific assay of citadiol--a key intermediate of escitalopram by liquid chromatography on Chiralpak AD-H column connected with UV and polarimetric detectors in series

A simple, rapid, selective and reproducible LC method for separation and quantitative determination of citadiol (CTD), a key intermediate of escitalopram has been developed. An optimum resolution > 3.0 was achieved on Chiralpak AD-H (250 mm x 4.6 mm); 5 microm column connected with UV and polarimetric detectors in series. The effects of organic modifiers, viz., methanol, ethanol, n-propanol and 2-propanol on enantioselectivity were evaluated. The limits of detection (LOD) and quantification (LOQ) were 0.02 microg/ml, 0.03 microg/ml and 0.07 microg/ml, 0.10 microg/ml for R-CTD and S-CTD enantiomers, respectively. The linearity of the method was studied in the range of 0.07-300 microg/ml and 0.1-300 microg/ml for R-CTD and S-CTD, respectively and the r2 was > or = 0.9999. The inter- and intra-day assay precision was less than 0.74% (%R.S.D.) and the recoveries were in the range 99.68-100.72% with %R.S.D. < 0.49%.     

High performance liquid chromatographic determination of a new antifungal compound, ADKZ in rat plasma

A high performance liquid chromatography (HPLC) method was developed and validated for the determination of ADKZ (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl) -3-[N-methyl-N-(4-iodo-benzyl)amino]-2-propanol) in rat plasma. The compound was extracted from plasma samples by liquid-liquid extraction, and an isomeric compound of ADKZ (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[N-methyl-N -(3-iodo-benzyl)amino]-2-propanol) was used as the internal standard (IS), which were analyzed on a reversed-phase C18 column (5 microm, 200 mm x 4.6 mm i.d.). The extracted plasma samples were eluted with acetonitrile-0.018 M triethylamine solution adjusted to pH 3.2 with phosphoric acid (35:65, v/v). The effluent was monitored by a UV detector at 230 nm. The retention time of ADKZ was 7.1 min and IS 8.2 min. The calibration curves were linear in the concentration range of 0.02-2.00 microg/ml with the correlation coefficients greater than 0.999. The quantification limit of ADKZ in rat plasma was 0.02 microg/ml. Intra- and inter-day precision ranged from 2.6 to 7.9% and 3.1 to 9.6%, respectively. The extraction recovery from plasma was no less than 80%. No endogenous interferences were observed with either ADKZ or IS. The method has been successfully used to support the pre-clinical pharmacokinetic studies of ADKZ in rats.     

Simultaneous determination of zonisamide, carbamazepine and carbamazepine-10,11-epoxide in infant serum by high-performance liquid chromatography

This study developed a simple method for the simultaneous determination of zonisamide (ZNS), carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZE) in infant serum using reversed-phase high-performance liquid chromatograph (HPLC). The method involves a single-step protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C18 column (3 microm, 4.6 mm x 150 mm) with potassium phosphate buffer (pH 4.6; 25 mM)-methanol-acetonitrile (65:20:15 (v/v/v)) as a mobile phase at a 1.0 ml/min flow rate: ZNS was detectable using a UV detector at 235 nm, and both CBZ and CBZE were at 215 nm. The quantification limits were established in accordance with each therapeutic range at 2.5 microg/ml for ZNS, 0.5 microg/ml for CBZ, and 0.25 microg/ml for CBZE. The respective coefficients of variation were 1.3-6.0% and 2.2-7.7% for the intra- and inter-assay.     

Enantiomeric separation of cizolirtine and metabolites on amylose tris (3,5-dimethylphenyl carbamate) chiral stationary phase

The enantiomeric resolution of (+/-)-cizolirtine, (+/-)-cizolirtine-N-oxide, (+/-)-N-desmethylcizolirtine and (+/-)-5(alpha-hydroxybenzyl)-1-methylpyrazole was achieved on amylose tris (3,5-dimethylphenyl carbamate) chiral stationary phase known as Chiralpak AD using hexane/2-propanol/triethylamine (80:20:0.05, v/v/v) as the mobile phase. The flow rate of the mobile phase used was 1.0 ml/min with UV detection at 230 nm. The values of R(s) of the resolved enantiomers of (+/-)-cizolirtine, (+/-)-cizolirtine-N-oxide, (+/-)-N-desmethylcizolirtine and (+/-)-5(alpha-hydroxybenzyl)-1-methyl pyrazole were 1.20, 0.60, 1.16 and 1.15, respectively.     

A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.     

HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment

A novel fast isocratic reversed-phase HPLC method for simultaneous determination of chlorhexidine and its degradation product p-chloroaniline was developed. Zorbax SB Phenyl column (75 mm x 4.6 mm, 3.5 microm) was used for the separation. Mobile phase composed of acetonitrile and buffer solution of 0.08 M sodium phosphate monobasic containing 5 ml of triethylamine (0.5%) and adjust with 85% phosphoric acid to pH 3.0 in ratio 35:65 (v/v) pumped isocratically at flow rate 0.6 ml min(-1) was used. UV detection was performed at 239 nm, the total analysis time was about 10 min. The method is suitable for practical routine analysis of topical ointment in the quality control laboratory.     

A liquid chromatographic method for simultaneous determination of amoxicillin sodium and sulbactam sodium in a combination formulation

An isocratic liquid chromatographic method with UV detection at 210nm is described for simultaneous determination of amoxicillin sodium and sulbactam sodium in a new combination formulation. Chromatographic separation of the two drugs was achieved on a Hypersil C(18) column using a mobile phase consisting of a binary mixture of methanol and 0.01mol/l sodium acetate (5:95, v/v). The commonly used paired-ion aqueous mobile phase for the determination of penicillins was avoided in this study. The developed LC method offers symmetric peak shape, good resolution and reasonable retention time for both drugs. Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 155.3-1553.0microg/ml for amoxicillin sodium and 45.0-450.0microg/ml for sulbactam sodium. The proposed LC method can be used for the quality control of formulated products containing these two drugs.     

A simple and rapid high-performance liquid chromatographic method for the determination of bisoprolol fumarate and hydrochlorothiazide in a tablet dosage form

A simple and precise high performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate (BF), and hydrochlorothiazide (HCTZ) in a tablet formulation. Chromatography was carried out at 25 degrees C on a 4.6 mm x 250 mm, 5 microm cyano column with the isocratic mobile phase of 0.1M aqueous phosphate buffer, acetonitrile and tetrahydrofuran (85:10:5, v/v/v) at a flow rate of 1.0 ml/min. The UV detection was carried out at 225 nm. HCTZ and BF were separated in less than 10 min with good resolution and minimal tailing, without interference of excipients. The method was validated according to ICH guidelines and the acceptance criteria for accuracy, precision, linearity, specificity and system suitability were met in all cases. The method was linear in the range of 50-150 microg/ml for BF and 125-375 microg/ml for HCTZ.     

Determination of cetylpyridinium chloride and tetracaine hydrochloride in buccal tablets by RP-HPLC

The HPLC method for simultaneous determination of cetylpyridinium chloride (CPC), tetracaine hydrochloride (TTC) in Xipiluan buccal tablets was developed and validated. The HPLC method was performed on a CN column (150 x 4.6 mm i.d., 5 microm particle size); the mobile phase was methanol-tetramethylammonium hydroxide (20 mM)-potassium dihydrogen phosphate (3 mM) (90:10:3, v/v/v) (pH* 5.0), pumped at a flow rate 1.5 ml min(-1). The UV detector was set at 230 nm. The retention time for CPC and TTC was 3.52 and 3.10 min, respectively. Calibration curves were linear (r=0.9999, n=6) in the range of 5-2000 microg ml(-1) for CPC and 1-500 microg ml(-1) for TTC. Limit of detection and quantitation for CPC was 0.033 and 0.11 microg ml(-1), for TTC were 0.0056 and 0.019 microg ml(-1). The R.S.D. of repeatability and intermediate precision for CPC and TTC were less than 2.0%.     

Enantiomeric resolution of doxazosin mesylate and its process-related substances on polysaccharide chiral stationary phases

High-performance liquid chromatographic methods for separation of racemic doxazosin mesylate and its synthetic precursors on polysaccharide based stationary phases viz., amylose tris-(3,5-dimethylphenylcarbamate) (Chiralpak AD-H) and cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) were developed. The base line separation with Rs>1.50 was obtained using a mobile phase containing n-hexane-alcohol-0.1% diethylamine (ethanol, 1-propanol and 2-propanol) in various proportions. The effect of concentration of the alcoholic modifiers on the resolution was studied. A good separation was achieved on amylose based Chiralpak AD-H column when compared with cellulose based Chiralcel OD-H. The effects of structural features of the solutes and solvents on discrimination between the enantiomers were examined. The detection was carried out at 240 nm with UV detector while identification by polarimetric detector connected in series. The method was suitable not only for process development of doxazosin mesylate but also determination of enantiomeric purity of bulk drugs and pharmaceuticals.     

Simultaneous determination of hydrochlorothiazide and amiloride hydrochloride by ratio spectra derivative spectrophotometry and high-performance liquid chromatography

Rapid, precise, accurate and specific ratio spectra derivative spectrophotometry and high-performance liquid chromatographic procedures were described for the simultaneous determination of hydrochlorothiazide and amiloride hydrochloride in combined pharmaceutical dosage forms. For the first method, ratio spectra derivative spectrophotometry, the signals were measured at 285.7 nm for hydrochlorothiazide and at 302.5 nm for amiloride hydrochloride in the mixture, in the first derivative of the ratio spectra. The second method is based on high-performance liquid chromatography (HPLC) on LiChrosorb RP-C18 column (5 microm, 20 cm x 4.6 mm) using 0.025 M orthophosphoric acid (adjusted to pH 3.0 with triethylamine (TEA)), acetonitrile (84:16 v/v) as a mobile phase at a flow rate of 1.2 ml/min(-1). Detection was carried out using a UV detector at 278.0 nm. Commercial sugar-coated and laboratory-prepared mixtures containing both drugs in different proportions were assayed using the developed methods.     

Stereoselective determination of pyridoglutethimide enantiomers in serum with a chiral cellulose-based high-performance liquid chromatographic column using solid phase extraction and UV detection

A sensitive method for the separation and determination of R(+)- and S(-) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for R(+)- and S(-)-pyridoglutethimide enantiomers were in the range 86-91% at 300-900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9-3.9 and 1.5-4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9-3.3 and 1.5-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100-1500 ng/ml for each enantiomer show correlation coefficient (r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (S/N = 2).     

LC determination of atropine sulfate and scopolamine hydrobromide in pharmaceuticals

An accurate, simple, reproducible and sensitive method for the determination of atropine sulfate and scopolamine hydrobromide has been developed and validated. Atropine sulfate and scopolamine hydrobromide were separated using a microBondapack C(18) column by isocratic elution with flow rate 1.0 ml/min. The mobile phase composition was methanol, water, formic acid (165:35:1; v/v/v) and pH adjusted 8.3 with triethylamine. The samples were detected at 230 nm using photo-diode array detector. The linear range of detection for atropine sulfate (I) and scopolamine hydrobromide (II) were between 10.38 and 1038 microg/ml with a limit of quantification (LOQ) of 10.38, 10.00 and 1034 microg/ml with an LOQ of 10.00 microg/ml respectively. The linearity, range, peak purity, selectivity, system performance parameters, precision, accuracy, robustness and ruggedness for (I) and (II) were also shown acceptable values.     

Simultaneous micellar LC determination of lidocaine and tolperisone

A micellar liquid chromatography (MLC) procedure was developed for the simultaneous separation and determination of lidocaine hydrochloride (LD HCl) and tolperisone hydrochloride (TP HCl) using a short-column C18 (12.5 mm x 4.6 mm, 5 microm), sodium dodecyl sulfate (SDS) with a small amount of isopropanol, and diode array detector. The optimum conditions for the simultaneous determination of both drugs were 0.075 mol l(-1) SDS-7.5% (v/v) isopropanol with a flow rate of 0.7 ml min(-1) and detection at 210 nm. The LOD (2S/N) of LD HCl was 0.73 ng 20 microl(-1), whereas that of TP HCl was 1.43 ng 20 microl(-1). The calibration curves for LD HCl and TP HCl were linear over the ranges 0.125-500 microg ml(-1) (r(2)=0.9999) and 1.00-500 microg ml(-1) (r(2)=0.9997), respectively. The %recoveries of both drugs were in the range 98-103% and the %RSD values were less than 2. The proposed method has been successfully applied to the simultaneous determination of TP HCl and LD HCl in various pharmaceutical preparations.     

Rapid and sensitive HPLC method for the simultaneous determination of dorzolamide hydrochloride and timolol maleate in eye drops with diode-array and UV detection

A rapid and sensitive HPLC method has been developed for the simultaneous determination of dorzolamide hydrochloride and timolol maleate. The drugs were monitored with a diode-array detector at two fixed wavelengths (lambda = 250.0 nm for dorzolamide hydrochloride and 300.0 nm for timolol maleate). Liquid chromatography was performed on a RP-YMC pack ODS A-132 C18 (5 microm, 15 cm x 6.0 mm) column and the mobile phase consisted of an acetonitrile: phosphate buffer (pH 2.5): methanol (5:85:10 v/v/v) mix and a flow rate of 1.2 ml x min(-1). The linearity of the method ranged between 4.0-45.0 microg x ml(-1) for dorzolamide hydrochloride and and 2.0-20.6 microg x ml(-1) for timolol maleate in binary mixture. The procedure was successfully applied to the determination of these compounds in pharmaceutical preparations and gave a high recovery, good accuracy and precision without any interference by the excipients.     

Quantitative determination of gatifloxacin, levofloxacin, lomefloxacin and pefloxacin fluoroquinolonic antibiotics in pharmaceutical preparations by high-performance liquid chromatography

The objective of this research was to develop and validate analytical methods for quantitative determination of fluoroquinolones of third generation. Simple and rapid chromatographic method was developed and validated for quantitative determination of four quinolone antibiotics in tablets and injection preparations. The fluoroquinolones studied were gatifloxacin (GAT), levofloxacin (LEV), lomefloxacin (LOM) and pefloxacin (PEF). The quinolones were analyzed by using a LiChrospher 100 RP-18 column (5 microm, 125 mm x 4 mm) and a mobile phase constituted of water:acetonitrile (80:20, v/v) with 0.3% of triethylamine and pH adjusted to 3.3 with phosphoric acid. The flow rate was 1.0 mL/min and the analyses were performed using UV detector with wavelengths varying from 279 to 295 nm. The analyses were performed at room temperature (24 +/- 2 degrees C). All fluoroquinolones were separated within 5 min. The calibration curves were linear (r>or=0.9999) over a concentration range from 4.0 to 24.0 microg/mL. The relative standard deviation (R.S.D.) was < 1.0% and average recovery was above 99.54%.