Development of a stacking-CZE method for the analysis of phenolic acids

Eight phenolic acids were analyzed by capillary zone electrophoresis. On-line analyte preconcentration was carried out by hydrodynamic injection of large volume of sample followed by removal of the bulk of the low conductivity sample matrix by polarity switching. The optimal electrolyte system consisted of 50mM sodium tetraborate of pH 9.0 (adjusted with 0.1 M phosphoric acid) containing 2% of alpha-cyclodextrin. The separations were carried out with a fused silica capillary (effective length 50 cm, i.d. 50 microm) and monitored at 200 nm. Under optimized preconcentration conditions (sample injection 99 s at 100 mbar and the polarity switching time 1.0 min) linear calibration ranges (0.1-2.0 microg/ml, R=0.9979-0.9995), favourable limits of detection (0.01-0.025 microg/ml) and good repeatability of the peak areas (R.S.D.: 2.76-5.69%, n=6) were achieved.     

Development and validation of a high-resolution capillary electrophoresis method for multi-analysis of ragaglitazar and arginine in active pharmaceutical ingredients and low-dose tablets

A selective, sensitive and robust capillary electrophoresis (CE) method has been developed and validated for multi analysis of ragaglitazar (also known as NNC 61-0029 or DRF 2725) and its counter ion arginine in Active Pharmaceutical Ingredients (API) and low-dose tablets (0.5, 1.0 and 2.0 mg). The method covers a total number of 12 analyses for the API and tablets: assay and identification of ragaglitazar and arginine, chiral purity of ragaglitazar and purity of ragaglitazar. After a simple extraction of samples with acetonitrile and 0.01 M sodium hydroxide (10:90), separation was performed using a combination of two cyclodextrins; sulfobutylether-beta-cyclodextrin (SB-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) in the electrolyte. The method showed good specificity and could separate and detect ragaglitazar, the distomer (the (+)-enantiomer) and arginine. The LOQ was found to be 0.10%, corresponding to 0.2 ng (0.5 microg/ml) for ragaglitazar and the distomer. Linearity was observed to be from 0.5 to 15 microg/ml (range 0.2-60 ng) and 400-600 microg/ml (range 1603-2404 ng) for ragaglitazar and 166-250 microg/ml (range 668-1000 ng) for arginine. The accuracy (as percent recovery) for ragaglitazar was found to be 101-106% (at 400-600 microg/ml), 101-125% (at 0.5-15 microg/ml) and for arginine 97-101% (at 166-250 microg/ml). The repeatability for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.05%, distomer: 1.01%, largest single impurity: 5.84%, total impurities: 0.90% and arginine: 2.00%. The intermediate precision for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.63%, distomer: 1.98%, largest single impurity: 5.22%, total impurities: 13.17% and arginine: 3.50.     

Development and validation of an assay for citric acid/citrate and phosphate in pharmaceutical dosage forms using ion chromatography with suppressed conductivity detection

The paper describes the development and validation of a simple, rapid, accurate, and sensitive ion chromatographic procedure to assay total citrate (citric acid/citrate) and phosphate in nine dosage forms. The dosage forms chosen represent all dosage forms in USP27-NF22 for which the respective monographs require an assay for either citric acid/citrate or citric acid/citrate and phosphate. Citrate and phosphate were separated in <10min by a hydroxide-selective column using anion-exchange chromatography with a 20mM potassium hydroxide eluent and detected by suppressed conductivity. The method showed linear responses over the concentration ranges 0.2-100microg ml(-1) (r(2) > 0.9990) for citrate and 0.2-60microg ml(-1) (r(2) = 0.9999) for phosphate, with limits of quantitation (signal-to-noise (S/N) = 10) of 0.2microg ml(-1) for both analytes. The accuracy of the procedure, determined by spiked recovery measurements, was within 95-105%. The intraday and the interday precision were demonstrated by the relative standard deviations (R.S.D.) of <1 and <2%, respectively, for both analytes. The ruggedness was determined by a full factorial design using analyst, equipment, column lot, and eluent preparation procedure as variables. The results show an overall R.S.D. of <3% and that an electrolytically generated 20mM KOH eluent produces assay results equivalent to a manually prepared 20mM NaOH eluent.     

Determination of ursodeoxycholic acid in pharmaceutical preparations by capillary electrophoresis with indirect UV detection

A simple and rapid capillary electrophoretic method, with indirect UV detection, for the quantification of ursodeoxycholic acid (UDCA) in pharmaceutical preparations was developed in this study. Sodium p-hydroxy benzoate was used as background electrolyte (BGE) (5 mM, pH 8.0) and visualization agent. Separation was carried out on a fused-silica capillary (50 microm x 72 cm) at a potential of 25 kV under ambient temperature and detected at 250 nm. Glycocholic acid was used as internal standard for quantification. Both run-to-run repeatability and day-to-day reproducibility of migration time were below 0.1% relative standard deviation (R.S.D.). Repeatability and reproducibility of relative peak height were 3.3 and 3.8% R.S.D., respectively. Accuracy was tested by spiking two pharmaceutical tablets with standards and the recoveries were 101.9+/-9.87 and 99.6+/-9.60% (n=3), respectively. Linearity of relative peak height was tested in the range 20-100 microg/ml. Limit of detection (LOD) was 3 microg/ml. The method could be used to assay UDCA raw materials and formulation products.     

Preparation of chitosan beads by simultaneous cross-linking/insolubilisation in basic pH. Rheological optimisation and drug loading/release behaviour.

A one-step procedure to prepare chitosan beads by simultaneous cross-linking with glutaraldehyde and insolubilisation in 1.5 M NaOH solution has been developed. The optimisation of the procedure was carried out by monitoring the evolution of the loss and storage moduli of chitosan solutions (1.5% (w/v), in acetic acid 0.2 M) in the presence of different proportions of glutaraldehyde. Increasing the chitosan molecular weight, glutaraldehyde concentration and/or process temperature from 20 to 37 degrees C, a reduction of time to reach the gel point was observed. The diameter of freshly prepared swollen beads was 3.2+/-0.4 mm and, after drying 0.48+/-0.18 mm. Swollen or previously dried beads were loaded with metronidazole by immersion in 0.1% (w/v), drug solution in a phosphate buffer pH 7.5, purified water, 0.2 M acetic acid or 0.1 M HCl. Beads synthesised at 37 degrees C experimented faster swelling than the ones prepared at 20 degrees C and even disintegrated in acetic acid. The amounts of metronidazole loaded (ranging from 1 to 286 mg/g dried beads) increased with swelling capacity of beads. The release studies carried out in 0.1 M HCl indicated that, regardless of the medium used to load the beads, all of them released the dose in less than 30 min. In summary, applying this one-step procedure and choosing the adequate glutaraldehyde proportion, it is possible to obtain particles of chitosan cross-linked with itself, which exhibit pH-sensitive swelling and which are able to release all the drug quickly into an acidic environment such as the stomach. The results obtained also highlight the importance of the pH of the medium for modulating the amount of drug loaded (it is remarkably greater at lower pHs) and the influence of temperature at which the beads are prepared on their tendency to disintegrate.     

Fast and single solid phase fluorescence spectroscopic batch procedure for (acetyl) salicylic acid determination in drug formulations

A solid phase fluorescence spectroscopic batch procedure for (acetyl) salicylic acid in drug formulations have been developed. The procedure is based on the sorption of salicylic acid (SA) on Sephadex DEAE A-25 anion exchanger gel (100 mg) by equilibration from an aqueous solution (10 or 25 ml) for 5 min; the equilibrated gel is transferred into an 1 mm quartz cell and the native fluorescence of SA sorbed on it is directly measured (lambda(ex)=297 nm; lambda(em)=405 nm). Good linearity was found in the 10-200 and 5-100 microg l(-1) ranges (for 10 and 25 ml sample volume, respectively) with R.S.D. (%) of 2.8 and 1.1. The procedure was successfully applied to the determination of acetyl salicylic acid (ASA) in drug formulations after alkaline hydrolysis to yield SA.     

Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a clinical study

A simple, sensitive and specific LC-MS/MS method for simultaneous determination of rosuvastatin (RST) and fenofibric acid (FFA) was developed and validated with 500 microL human plasma using carbamazepine as an internal standard (IS). The assay procedure involved a simple one-step liquid/liquid extraction of RST and FFA and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto X-Terra MS C-18 column (4.6 mm x 50 mm, 5.0 microm). Separation of RST, FFA and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (45:55, v/v) at a flow rate of 0.40 ml/min. The API-3000LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Positive ion acquisition chromatographic run was used in the present method. Nominal retention times of RST, FFA and IS were 2.35, 4.70 and 2.32 min, respectively. Absolute recovery of RST, FFA and IS was 74, 61 and 69%, respectively. The lower limit of quantification (LLOQ) of RST and FFA was 1.00 ng/ml and 0.50 microg/ml, respectively. Response function was established for the range of concentrations 1.00-50.0 ng/ml and 0.50-20.0 microg/ml for RST and FFA, respectively, with a coefficient of determination (r2) of 0.999 for both the compounds. The inter- and intra-day precision in the measurement of RST quality control (QC) samples 5, 15, 400 and 800 ng/ml, were in the range 8.93-9.37% relative standard deviation (R.S.D.) and 1.74-16.1% R.S.D., respectively. Similarly, the inter- and intra-day precision in the measurement of FFA quality control (QC) samples 0.5, 1.5, 8.0 and 15.0 microg/ml, were in the range 9.78-11.6% relative standard deviation (R.S.D.) and 0.22-17.4% R.S.D., respectively. Accuracy in the measurement of QC samples for RST and FFA were in the range 88.1-108 and 87-115%, respectively, of the nominal values. RST and FFA were stable in the battery of stability studies, viz., bench-top, auto-sampler and freeze/thaw cycles. Stability of RST and FFA was established for 1 month at -80 degrees C. The application of the assay to a clinical study confirmed the utility of the assay.     

Quantitative determination of insulin entrapment efficiency in triblock copolymeric nanoparticles by high-performance liquid chromatography

A rapid and effective isocratic chromatographic procedure was described in this paper for the determination of insulin entrapment efficiency (EE) in triblock copolymeric nanoparticles using reversed-phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet/visible detector at low flow rate. The method has been developed on a Shimadzu Shim-pack VP-ODS column (150 mm x 4.6 mm, 5 microm, Chiyoda-Ku, Tokyo, Japan) using a mixture of 0.2 M sodium sulfate anhydrous solution adjusted to pH 2.3 with phosphoric acid and acetonitrile (73:27, v/v) as mobile phase at the flow rate of 0.8 ml min(-1) and a 214 nm detection. The method was validated in terms of selectivity, linearity, precision, accuracy, solution stability, limit of detection (LOD) and limit of quantification (LOQ). The calibration curve was linear in the concentration range of 2.0-500.0 microg ml(-1), and the limits of detection and quantitation were 8 and 20 ng, respectively. The mean recovery of insulin from spiked samples, in a concentration range of 8-100 microg ml(-1), was 98.96% (R.S.D.= 2.51%, n = 9). The intra- and inter-assay coefficients of variation were less than 2.24%. The proposed method has the advantages of simple pretreatment, rapid isolation, high specificity and precision, which can be used for direct analysis of insulin in commercially available raw materials, formulations of nanoparticles, and drug release as well as stability studies.     

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%.     

Simultaneous determinations of paeonol and palmatine hydrochloride in Shangshi Aerosols by HPLC method

A simple and rapid HPLC method was described for the simultaneous determination of paeonol and palmatine hydrochloride in Shangshi Aerosols. The optimum separation for these analytes was achieved using the mixture of 0.025 M sodium dihydrogen phosphate-acetonitrile-diethylamine (64:35:1, v/v/v) as the mobile phase and a Nova-Pak((R)) C8 column. The linear ranges of paeonol and palmatine hydrochloride were 0.2-80 and 0.06-60 microg/ml with the regression equations being Y=11716.4+2.96 x 10(6)X (R=0.99969), Y=-6388.8+1.89 x 10(5)X (R=0.99976), and limit of quantifications (LOQ) for paeonol and palmatine hydrochloride were 0.2 and 0.06 microg/ml, respectively (n=6). Other validation parameters: intra-day precision (R.S.D.: 0.71-1.65%) and inter-day precision (R.S.D.: 0.89-2.11%), and reproducibility (recoveries values: 94.6-98.2% for paeonol, 94.85-97.58% for palmatine hydrochloride) were found to be satisfactory. The proposed HPLC method had been applied for the determination of paeonol and palmatine hydrochloride in Shangshi Aerosols; R.S.D. values were 1.45 and 1.13%, respectively. In short, this method was rapid and convenient, which could be used for the routine control of paeonol and palmatine hydrochloride in Shangshi Aerosols.     

Stability study of the anticonvulsant enaminone (E118) using HPLC and LC-MS.

The stability of the new chemical synthetic enaminone derivative (E118) was investigated using a stability-indicating high-performance liquid chromatography (HPLC) procedure. The examined samples were analyzed using a chiral HSA column and a mobile phase (pH 7.5) containing n-octanoic acid (5 mM), isopropyl alcohol and 100 mM disodium hydrogen phosphate solution (1:9 v/v) at a flow rate of 1 ml min(-1). The developed method was specific, accurate and reproducible. The HPLC chromatograms exhibited well-resolved peaks of E118 and the degradation products at retention times <5 min. The stability of E118 was performed in 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, water/ethanol (1:1) and phosphate buffer (pH approximately 7.5) solutions. E118 was found to undergo fast hydrolysis in 0.1 M hydrochloric acid solution. The decomposition of E118 followed first order kinetics under the experimental conditions. The results confirmed that protonation of the enaminone system in the molecule enhanced the hydrolysis of E118 at degradation rate constant of 0.049 min(-1) and degradation half-life of 14.1 min at 25 degrees C. However, E118 was significantly stable in 0.1 M sodium hydroxide, physiological phosphate buffer (pH 7.5) and ethanol/water (1:1) solutions. The degradation rate constants and degradation half-lives were in the ranges 0.0023-0.0086 h(-1) and 80.6-150.6 h, respectively. Analysis of the acid-induced degraded solution of E118 by liquid chromatography-mass spectrometry (LC-MS) revealed at least two degradation products of E118 at m/z 213.1 and 113.1, respectively.     

FIA of sildenafil citrate using UV-detection

A flow injection analysis (FIA) of sildenafil citrate (SLD) using UV detection is described in this study. The best solvent system was found to be consisting of 0.2 M phosphate buffer at pH 8 having 10% MeOH. A flow rate of 1 ml. min(-1) was pumped and active material was detected at 292 nm. The calibration equation was linear in the range of 1x10(-6)-5x10(-6) M. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated to be 3x10(-7) and 8.9x10(-7) M with a R.S.D. 1.9 and 0.6% (n=7), respectively. The proposed method was applied to the determination of SLD in VIAGRA tablet, containing 50 mg active material. The results were compared with those obtained from UV-Spectrophotometry. The results showed that there is a good agreement between FIA method and the UV-Spectrophotometry. The validation studies were realised by the related applications and the results were evaluated statistically. According to the results, insignificant difference was observed between the methods.     

HPLC determination of sertraline in bulk drug, tablets and capsules using hydroxypropyl-beta-cyclodextrin as mobile phase additive

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for determination of sertraline in bulk drug, tablets and capsules was developed. Chromatography resolution of the sertraline enantiomeric forms and trans diastereoisomers was performed on Alltima C18 (250 mm x 4.6mm i.d., 5 microm) column with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as mobile phase additive. The composition of the mobile phase was 68:32 (v/v) aqueous 170 mM phosphate buffer, pH 3.0 (adjusted with 85% phosphoric acid) containing 18 mM HP-beta-CD/acetonitrile at a flow rate of 1.0 ml x min(-1). The UV detector was set at 225 nm. Calibration curves were linear (r=0.9999, n=9) in the range of 1-120 microgml (-1) for sertraline. Limit of detection and quantitation for sertraline was 0.029 and 0.097 microg x ml (-1). The values of R.S.D. of repeatability and intermediate precision for bulk drug, tablets and capsules of sertraline hydrochloride were less than 1.0%.     

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).     

Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection

Sonazoid is a new contrast agent for ultrasound imaging. The product is an aqueous suspension of perfluorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine (H-EPS). A normal-phase high-performance liquid chromatographic (HPLC) method with evaporative light scattering detection was developed for quantification of free fatty acids, phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid. Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 7.5 with ammonia. The calibration standards contained stearic acid, distearoyl-phosphatidic acid (DSPA) and distearoyl-phosphatidylserine (DSPS) in the concentration range of 0.016-1.0mg/ml (0.4-25microg injected). The method was validated with a limit of quantification of the three lipids set to 0.4microg (approximately 20-60microM). The best fit of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and fitted to a second order polynomial equation. Stearic acid, DSPA and DSPS were analysed with an intermediate precision ranging from 4.4% to 5.3% R.S.D. and they were extracted from an aqueous suspension with a recovery ranging from 103.3% to 113.3%. The sum of total phospholipid concentration determined in H-EPS ranged from 96.4% to 103.2% of the theoretical values. The lipids in the ultrasound product were quantitated with a repeatability ranging from 6.2% to 11.7% R.S.D.     

Application of nuclear magnetic resonance spectroscopy for quantitative analysis of miconazole, metronidazole and sulfamethoxazole in pharmaceutical and urine samples

Specific, accurate and precise NMR methods were developed for determining miconazole, metronidazole and sulfamethoxazole antibiotic drugs in authentic, pharmaceutical and urine samples. Proton nuclear magnetic resonance spectroscopy (1H NMR) with maleic acid as an internal standard and DMSO-d6 as NMR solvent were used. 1H NMR signals at 9.0, 8.06, 7.50 and 6.26 ppm corresponding to miconazole, metronidazole, sulfamethoxazole and maleic acid were respectively used for calculating the concentrations of drugs per unit dose. Average percent recoveries of (97.54-101.10), (98.06-100.46) and (97.83-102.83) with average uncertainties of 1.02, 0.45 and 0.86 were respectively obtained for determining authentic samples of miconazole, metronidazole and sulfamethoxazole in the concentration range of 0.92-170 mg/0.6 ml DMSO-d6. In pharmaceutical formulations and urine samples, average percent recoveries in the ranges of 97.50-101.33 and 94.46-100.86 were respectively obtained. Relative standard deviations (R.S.D.)相似文献   

Stir bar sorptive extraction with in situ de-conjugation and thermal desorption gas chromatography-mass spectrometry for measurement of 4-nonylphenol glucuronide in human urine sample

4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.     

Quantitative determination of amantadine in human plasma by liquid chromatography-mass spectrometry and the application in a bioequivalence study

A sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method is developed and validated for rapid determination of amantadine in human plasma. Desloratadine was used as the internal standard (I.S.). Human plasma (0.2 mL) was first alkalified with 100 microL of sodium hydroxide (3M) and then extracted with 1 mL of n-hexane containing 1% isopropanol (v/v) and 10% dichloromethane (v/v) by vortex-mixer for 3 min. The mixture was centrifuged at 14,000 rpm for 5 min. The supernatant was evaporated to dryness and the residue was dissolved in mobile phase. Samples were separated using a Thermo Hypersil-HyPURITYC18 reversed-phase column (150 mm x 2.1 mm i.d., 5 microm). Mobile phase consisted of methanol-acetonitrile-20 mM ammonium acetate (45:10:45, v/v/v) containing 1% acetic acid with pH 4.0. Amantadine and I.S. were measured by electrospray ion source in positive selective ion monitoring mode. The good linearity ranged from 3.9 to 1000 ng/mL and the lowest limit of quantification was 3.9 ng/mL. The extraction efficiencies were approximately 70% and recoveries of method ranged from 98.53 to 103.24%. The intra-day relative standard deviations (R.S.D.) were less than 8.43% and inter-day R.S.D. below 10.59%. The quality control samples were stable when kept at room temperature for 12h, at -20 degrees C for 30 days and after four freeze/thaw cycles. The method has been successfully used to evaluation of the pharmacokinetics and bioequivalence of amantadine in 20 healthy volunteers after an oral dose of 100 mg amantadine.     

Determination of verapamil by adsorptive stripping voltammetry in urine and pharmaceutical formulations

A sensitive reduction peak of verapamil is obtained by adsorptive stripping voltammetry in 0.01 M phosphate (pH 7.4) at an accumulation time of 30 s. The peak potential is -1.81 V (vs. Ag/AgCl). The peak current is directly proportional to the concentration of verapamil (1x10(-8)-1x10(-6) M), with a 3sigma detection limit of 5x10(-10) M (0.246 ng/ml). The R.S.D. at the 1x10(-7) M level is 1.8%. The interference of some metal ions, and some amino acids, and the application of the method to analysis of urine, and pharmaceutical formulations are described. The method is simple (no extraction), rapid (30 s accumulation time), sensitive (the detection limit of verapamil is 0.491 ng/ml), reproducible(within day R.S.D. of 1.28-1.8%), and suitable for routine analysis of verapamil, urine, and pharmaceutical formulation.     

Direct chiral separation of troglitazone stereoisomers using reversed-phase high-performance liquid chromatography

A simple HPLC method for the direct chiral separation of troglitazone stereoisomers was developed. The separation was performed on a reversed-phase cellulose-derivertized chiral column (Chiralcel OJ-R) using a mobile phase consisting of methanol-acetic acid (1000:1, v/v) at a flow rate of 0.5 ml/min. The peak areas of stereoisomers separated from 0.13 to 0.75 mg/ml of troglitazone had good linearity, with correlation coefficients > 0.999 in the reversed-phase mode. The repeatability of the ratios of stereoisomers isolated from 0.5 mg/ml of troglitazone had a relative standard deviation of 0.1-0.2%. The relative sensitivities of the four isomers at UV 285 nm were similar, as each response factor was within the range of 0.99-1.01. Troglitazone racemized at the chiral center of the thiazolidine ring in methanol solution, but was found to be stable for 24 h in methanol-acetic acid (1000:1, v/v). This method was applied to the stereoisomeric analysis of troglitazone in pharmaceutical formulations and used to evaluate the constancy of the stereoisomer ratio in the manufacturing process and stability testing.