A Validated, Stability-Indicating, LC Method for Rabeprazole Sodium

We present a simple and reliable method for simultaneous determination of voriconazole and its main metabolite resulting from N-oxidation (UK-121,265), in human plasma. The work-up procedure used acetonitrile and potassium salts to precipitate plasma proteins. No internal standard was used. The chromatographic system used a LiChroCART^® 250-4 cartridge packed with LiChrospher^® 100 RP-8 (diameter particules, 5 μm). The UV monochromatic detector was set on 260 nm. The mobile phase consisted of a 60/40 (v/v) mixture of acetonitrile and water. The flow rate was 1 mL min^?1. The retention times for voriconazole and its metabolite were 8.98 and 4.02 min respectively, and total run time was 12 min. The linearity of the method was investigated from 0.31 to 10.0 mg L^?1; the lowest limit of quantification was 0.30 mg L^?1. Precision ranged from 2.41% to 6.32% for voriconazole and 0.80% to 11.6% for the N-oxide voriconazole metabolite. Accuracy was between 93.0% and 101% for voriconazole and 90.0% and 101% for the N-oxide voriconazole metabolite. This rapid and accurate method could be interesting to investigate metabolite/voriconazole ratio with respect to CYP2C19 genetic status and CYP3A4 activity changes.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.     

Selective LC Determination of Cabergoline in the Bulk Drug and in Tablets: In Vitro Dissolution Studies

Cabergoline (CAB) is an ergot alkaloid derivative with dopamine agonist activity. A novel, simple, and rapid stability-indicating high-performance liquid chromatographic (HPLC) method for assay of CAB in tablets has been developed and validated. Chromatography was performed on a 4.6 mm i.d. × 250 mm, 5 μm particle, cyano column with acetonitrile–10 mM phosphoric acid, 35:65 (v/v), containing 0.04% triethylamine, as mobile phase, at a flow rate of 1.0 mL min^?1, and UV detection at 280 nm. Response was a linear function of concentration in the range 0.1–4 μg mL^?1 (r ² = 0.9999). The recovery of the method was good (99.45%) and RSD values for intra-day and inter-day precision were 0.24–0.88% and 0.66–1.19%, respectively. The method can be used for quality-control assay of CAB in tablets, for stability studies, and for in vitro dissolution studies.     

LC Assay of Eletriptan in Tablets and In Vitro Dissolution Studies

Eletriptan (ELT) is a new selective serotonin agonist approved for the treatment of acute migraine headaches. A simple and rapid liquid chromatographic method was developed and validated for the assay of ELT in tablets. Chromatography was carried out on a 250 mm × 4.6 mm C₁₈ column at 30 °C. Acetonitrile–15 mM triethylamine solution (adjusted to pH 7.0 using concentrated o-phosphoric acid) (60:40, v/v) mixture was used as mobile phase at 1.0 mL min^?1 flow rate and UV detector was set at 225 nm. A linear response (r ²  = 0.9999) was observed in the range of 0.1–1.6 μg mL^?1. The method showed good recoveries (100.08 %) and the RSD values for intra- and inter-day precision were 0.78–1.93 and 1.10–2.15%, respectively. The method can be used for quality control assays and in vitro dissolution studies of ELT in tablets.     

Determination of Levamisole in Sheep Muscle Tissue by High-Performance Liquid Chromatography and Photo Diode Array Detection

A high-performance liquid chromatographic method for the determination of levamisole (LVM) residues in sheep muscle tissue is described. LVM was extracted with ethyl acetate under alkaline conditions and cleanup was performed by liquid-liquid partition between organic-basic and organic-acid medium. Finally, levamisole was back extracted with chloroform carefully transferred into a clean glass vial and evaporated to dryness at 50 °C under a gentle stream of nitrogen. The remaining dry residue was dissolved in the mobile phase used, filtered and an aliquot was injected automatically into the chromatograph for analysis. Chromatography was performed on a Zorbax^?SB-C₁₈ column at 50 °C and detection by a PDA detector monitored at λ_max 220 nm. The mobile phase was a mixture of 0.1 % trifluoroacetic acid (v/v) pH 2.0 and acetonitrile-methanol 3 : 2 (v/v) in a combination of 30 : 70 (v/v) and a flow rate of 1.0 mL min⁻¹, delivered isocratically. This analytical method was validated by assessing recovery efficiency using spiked muscle tissue samples with standard solutions in methanol at four fortification levels of 1/2 MRL, 1 MRL, 2 MRL and 4 MRL and five times for each concentration (n = 5). Mean recovery (R%) achieved for muscle tissue was 75.65 ± 2.74% with an acceptable Relative Standard Deviation RSD% = 10.4. The same method was used also for the analysis of kidney, liver and fat (perirenal) and the recoveries found were 70.25 ± 1.07% (RSD% = 1.52), 72.37 ± 3.6% (RSD% = 4.97) and 69.44 ± 2.22% (RSD% = 3.19), respectively. The limit of detection (LOD) for muscle tissue was found to be 2.0 μg kg⁻¹ and the limit of quantification (LOQ) 5.0 μg kg⁻¹. Revised: 4 and 24 January 2006     

Simultaneous Estimation of Atorvastatin Calcium, Ramipril and Aspirin in Capsule Dosage Form by RP-LC

A simple, sensitive, precise and accurate reversed phase liquid chromatographic method has been developed for the simultaneous estimation of atorvastatin (AT) calcium, ramipril (RA) and aspirin (AS) from capsule dosage form. The method was developed using a Phenomenex Luna C₁₈ (250 mm, 4.6 mm i.d., 5 µm) column with a mobile phase consisting of 0.1%, orthophosphoric acid buffer:acetonitrile:methanol (45:50:5 v/v/v), pH 3.3, at a flow rate of 1 mL min^?1. Detection was carried out with ultra-violet detection at 210 nm. The retention times were about 12.19, 2.35, and 3.95 min for AT calcium, RA and AS, respectively. The developed method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linearity ranges were 1–6 µg mL^?1 for AT calcium, 0.5–3 µg mL^?1 for RA and 7.5–45 µg mL^?1 for AS with mean recoveries of 100.59 ± 0.68, 100.62 ± 0.83 and 100.49 ± 0.73% for AT calcium, RA and AS, respectively. Limit of detection obtained were 29.85 ng mL^?1 for AT calcium, 4.71 ng mL^?1 for RA and 85.13 ng mL^?1 for AS. Impurity of salicylic acid was found in capsule dosage form at the retention time of about 4.84 min. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

High-Performance Liquid Chromatographic Determination of Isofraxidin in Rat Plasma

For the first time a high-performance liquid chromatographic (HPLC) method, with liquid-liquid extraction and ultraviolet (UV) absorbance detection, has been developed for quantification of isofraxidin in rat plasma. The analysis was performed on a Diamonsil C₁₈ column (200 mm × 4.6 mm i.d., 5 μm particle size) with acetonitrile–0.05% phosphoric acid, 26:74 (v/v), as isocratic mobile phase. The linear range was 0.05–8.0 μg mL⁻¹ and the lower limit of quantification was 0.05 μg mL⁻¹. The intra and inter-day relative standard deviation (RSD) for measurement of 0.25, 2.0, and 6.0 μg mL⁻¹ quality-control (QC) samples ranged from 5.7 to 6.4% and from 6.3 to 7.9%, respectively. Accuracy, as relative error (RE), was from ±5.8% to ±7.3%. The method was validated for specificity, accuracy, and precision and was successfully used in a pharmacokinetic study of isofraxidin in rat plasma after administration of Ciwujia extract.     

An Improved UPLC Method for Rapid Analysis of Levofloxacin in Human Plasma

A rapid, specific, and sensitive ultra-performance liquid chromatographic method for analysis of levofloxacin in human plasma has been developed and validated. Plasma samples were spiked with the internal standard (enoxacin) and extracted with 10:1 (v/v) ethyl acetate–isopropanol. UPLC was performed on a 100 × 2.1 mm i.d., 1.7 µm particle, C₁₈ column with 88:12 (v/v) 0.4% triethylamine buffer (pH 3)–acetonitrile as mobile phase, pumped isocratically at a pressure of 11,000 psi (758 bar) and a flow-rate of 0.3 mL min^?1. Ultraviolet detection was performed at 300 nm. The retention times of levofloxacin and enoxacin were 3.4 and 2.8 min, respectively, and the run-time was 5 min. Calibration showed that response was a linear function of concentration over the range 0.05–10 µg mL^?1 (r ² ≥ 0.99) and the method was validated over this range for both precision and accuracy. The relative standard deviation was <15% for both intra-day and inter-day assay (n = 5). Levofloxacin and enoxacin were stable in plasma; there was no evidence of degradation during three freeze–thaw cycles, post-preparative stability at 20 °C was ≥24 h, short-term stability at room temperature was ≥6 h, and long-term stability at ?70 °C was ≥30 days. The method was successfully used in a study of the bioequivalence of two levofloxacin tablet formulations in healthy volunteers.     

Chemometrically Assisted Development and Validation of LC for Simultaneous Determination of Carbamazepine and Its Impurities Iminostilbene and Iminodibenzyl in Solid Dosage Form

A chemometrical approach was applied to develop a reversed-phase liquid chromatographic method for simultaneous determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form. According to contemporary literature, no method was developed for simultaneous determination of carbamazepine and these impurities by chemometrical approach. The fractional factorial design was used for selection of variables significantly influencing the chromatographic separation of the investigated substances. The investigated variables were: temperature of the column, the percentage of organic modifier, the acetate buffer concentration and pH of water phase. The first three variables were proved to be significant and were optimized by face centered, central composite design. Investigation was performed using C18 XBridge Shield analytical column (50 mm × 4.6 mm i.d., particle size 3.5 µm). The optimal conditions for the separation were established with the mobile phase composition of methanol–10 mM acetate buffer (pH adjusted to 2.21 with glacial acetic acid) (50:50, v/v) at a flow rate of 1.5 mL min^?1, 25 °C column temperature and detection at 260 nm. Total analysis time was shortened to about 8 min. Finally, the method was successfully validated and subsequently applied to the analysis of commercially available carbamazepine tablets.     

Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection

A simple method has been developed for analysis of ibandronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPIC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions satisfactory separation of the compounds was achieved on an Intersil C₈ column with an isocratic mobile phase—8:4:88 (v/v) acetonitrile–methanol–12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile phase flow rate was 1.0 mL min^?1. The calibration plot was linear in the range 352 to 1760 µg mL^?1 for ibandronate. The precision and reproducibility were 0.3% and 0.5%, respectively. The average recovery of ibandronate was 100.4% and RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for assay of ibandronate in bulk material and dosage forms. The proposed liquid chromatographic method can be satisfactorily used for quality control of ibandronate.     

Determination of Carvedilol and its Impurities in Pharmaceuticals

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for separation of carvedilol and its impurities from Karvileks tablets. The best separation was achieved on a 100 mm × 4.6 mm, 5 µm particle size, Chromolit RP 8e column. Use of acetonitrile-water, 45:55 (v/v), adjusted to pH 2.5 with formic acid, as mobile phase at a flow rate of 0.5 mL min^?1 enabled acceptable resolution of carvedilol, in large excess, from possible impurities, in a short elution time. UV detection was performed at 280 nm. Linearity, accuracy, precision, selectivity, and robustness were validated and found to be satisfactory. Overall, the proposed method was found to be highly sensitive, suitable, and accurate for quantitative determination of carvedilol and its impurities in dosage forms and in raw materials.     

Improving the Determination of Nitrovin in Feeds by Reversed-Phase LC with SPE

A simple reversed-phase liquid chromatographic method with ultraviolet detector (378 nm) for the determination of nitrovin in feeds was improved and validated. The mobile phase was a mixture of acetonitrile and 0.1% formic acid solution (v/v) in the ratio of 50:50 (v/v), and the flow rate was set at 1.2 mL min^?1. The extraction solution was a mixture of dimethyl formamide, acetonitrile and methanol (50:25:25, v/v), the sample was cleaned-up with reversed-phase solid phase extraction cartridge. The standard nitrovin was purified with crude nitrovin product by ethylene glycol monoethyl ether and identified by elemental analyzer. The limit of detection was 0.05 mg kg^?1 and the limit of quatification was 0.2 mg kg^?1 in feeds. The assay had satisfactory selectivity, recovery, linearity and precise repeatability and trueness.     

HPLC Determination and Pharmacokinetic Study of Indapamide in Human Whole Blood

A rapid and sensitive HPLC method for the quantification of indapamide in human whole blood is described. The procedure involves liquid–liquid extraction of human whole blood with methyl tertiarybutyl ether coupled with reversed-phase HPLC and UV detection. Separation was performed on a YMC^® ODS-A reverse column (5 µm particle size, 4.6×150 mm i.d.). The mobile phase was composed of acetonitrile - 2-propanol ?0.1 triethylamine in water (adjusting to pH 3.75 with 85% phosphoric acid) (35:5:60, v/v/v). The linear concentration range for indapamide was 5.0–500 ngmL^?1. The lower limit of quantification (LLOQ) was 5.0 ngmL^?1 for indapamide. Intra- and inter-assay RSD ranged from 2.36% to 4.53% and 1.68% to 8.01%, respectively. The sensitivity and precision were sufficient for determination of whole blood concentrations after therapeutic administration of both drugs and the method can be used for the estimation of pharmacokinetic parameters.     

Development and Validation of a Reversed-Phase HPLC Method for the Determination of Deflazacort in Pharmaceutical Dosage Forms

A reversed-phase high-performance liquid chromatographic method has been developed and validated for quantitative determination of deflazacort in tablets and in compounded capsules. Isocratic chromatography was performed on a C₁₈ column with acetonitrile–water 80:20 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. UV detection was at 240 nm. The linearity of the method was good (r > 0.999), as also were intra-day and inter-day precision (RSD <2%) and accuracy (recovery >98%). The method was also validated for specificity and robustness. The results showed the proposed method is suitable for its intended use.     

Optimization,Validation and Application of a Capillary Zone Electrophoresis Method for the Assay of Sparfloxacin in Pharmaceutical Formulation

Abstract

A capillary zone electrophoresis (CZE) method has been developed for the determination of the antibiotic sparfloxacin in tablets. The CZE separation was performed using 75 µm×35 cm fused-silica capillary under the following conditions: 25°C; applied voltage, 12 kV; 25 mM H₃PO₄-NaOH running buffer (pH 8.5). The detection wavelength was 254 nm. Flumequine was used as internal standard (IS). The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision, specificity, and robustness. The calibration was linear from 10 to 60 µg mL^?1 and the limit of detection and quantification were 5.38 and 9.46 µg mL^?1, respectively. Recoveries ranging from 95.68%–102.4% were obtained in the determination of sparfloxacin that were spiked to placebos. Excipients in the commercial tablets and degraded products from different stress conditions did not interfere in the assay. The method was successfully applied to the determination of sparfloxacin in pharmaceutical tablets.     

Quantitative Analysis of Busulfan in Human Plasma by LC-MS–MS

High-performance liquid chromatography with tandem mass spectrometry has been used for rapid, specific, and sensitive analysis of busulfan in human plasma. Busulfan-d₈ was used as internal standard. Analysis was performed on a C₁₈ column (50 mm × 2.1 mm, 3.5-µm particles) with water–methanol 80:20 (v/v) as mobile phase at a flow-rate of 0.30 mL min^?1. Detection was by tandem triple–quadrupole mass spectrometry with turbo ion-spray ionization. Linear calibration plots were obtained over the concentration range 1.096–1,096 ng mL^?1. The assay is ideally suited to monitoring of busulfan and determination of its pharmacokinetic data.     

Determination of Veterinary Pharmaceuticals in Production Wastewater by HPTLC-Videodensitometry

An SPE-HPTLC method for simultaneous identification and quantification of seven pharmaceuticals in production wastewater was optimized and validated. The studied compounds were enrofloxacine, oxytetracycline, trimethoprim, sulfamethazine, sulfadiazine, sulfaguanidine and penicillin G/procaine. The method involves solid-phase extraction on hydrophilic-lipophilic balance cartridges with methanol and HPTLC analysis of extracts on CN modified chromatographic plates followed by videodensitometry at 254 and 366 nm. Optimization of chromatographic separation was performed by systematic variation of the mobile phase composition using genetic algorithm approach and the optimum mobile phase composition for TLC separation was 0.05 M H₂C₂O₄:methanol = 0.81:0.19 (v/v). Linearity of the method was demonstrated in the ranges from 1.5 to 15.0 μg L⁻¹ for enrofloxacine, 100–500 μg L⁻¹ for oxytetracycline, 150–600 μg L⁻¹ for trimethoprim, 300–1100 μg L⁻¹ for sulfaguanidine and 100–400 μg L⁻¹ for sulfamethazine, sulfadiazine and penicillin G/procaine with coefficients of determination higher than 0.991. Mean recoveries ranged from 74.6 to 117.1% and 55.1 to 108.0% for wellspring water and production wastewater, respectively. Only sulfaguanidine showed lower results. The described method has been applied to the determination of pharmaceuticals in wastewater samples from pharmaceutical industry.     

Stability-Indicating RP-HPLC Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in Dosage Forms

A new stability-indicating high-performance liquid chromatographic method has been developed for simultaneous analysis of metformin hydrochloride (MET) and sitagliptin phosphate (SIT) in pharmaceutical dosage forms. Chromatographic separation was achieved on a C₈ column. The mobile phase was methanol–water 45:55 % (v/v) containing 0.2 % (w/v) n-heptanesulfonic acid and 0.2 % (v/v) triethylamine; the pH was adjusted to 3.0 with orthophosphoric acid. The flow rate was 1 mL min^?1 and the photodiode-array detection wavelength was 267 nm. The linear regression coefficients for metformin and sitagliptin were 0.9998 and 0.9996 in the concentration ranges 50–450, and 10–150 μg mL^?1, respectively. The relative standard deviations for intra and inter-day precision were below 1.5 %. The drugs were subjected to a variety of stress conditions—acidic and basic hydrolysis, and oxidative, photolytic, neutral, and thermal degradation. The products obtained from photolytic degradation were similar to those from neutral hydrolytic degradation and different from produced by acidic and basic hydrolysis. The method resulted in detection of 15 degradation products (D1–D15); among these, the structures of D1, D3, D9, and D13 were identified. The respective mass balance for MET and SIT was found to be close to 97.60 and 99.12 %. The specificity of the method is suitable for a stability-indicating assay.     

Development and Validation of an RP-HPLC Method for Determination of Valganciclovir in Human Serum and Tablets

This paper describes the validation of an isocratic high-performance liquid chromatographic method for the assay of valganciclovir in raw materials, tablets and human serum samples. Valganciclovir and fluvastatin (internal standard) were well separated using a reversed phase column and a mobile phase consisting of a mixture of acetonitrile:methanol:KH₂PO₄ (0.02 M) (40:20:40; v/v/v) (at pH 5.0). The mobile phase was pumped at 1.0 mL min⁻¹ flow rate and valganciclovir was detected by diode-array detection at 255 nm. The retention times for valganciclovir and fluvastatin were 3.41 and 5.60 min, respectively. A linear response (r > 0.999) was observed in the range of 10–30,000 ng mL⁻¹ in mobile phase and serum. The limit of detection and limit of quantification were found as 2.95 and 9.82 ng mL⁻¹ in mobile phase and 1.73 and 5.77 ng mL⁻¹ in human serum samples, respectively. Validation parameters as precision, accuracy, selectivity, reproducibility and system suitability tests were also determined. The method can be used for valganciclovir assay of tablets and human serum samples as the method separates valganciclovir from tablet excipients and endogenous substances.

     

Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend^®) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min⁻¹. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and p^H; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and p^H have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control.