Ultra high-pressure liquid chromatographic assay of moxifloxacin in rabbit aqueous humor after topical instillation of moxifloxacin nanoparticles

The present report describes a rapid and sensitive ultra high-pressure liquid chromatography (UHPLC) method with UV detection to quantify moxifloxacin in rabbit aqueous humor. After deproteinisation with acetonitrile, gradient separation of moxifloxacin was achieved on a Waters Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm) column at 50 °C. The mobile phase consisted of 0.1% trifluoroacetic acid in water and acetonitrile at a flow rate of 0.4 ml/min. Detection of moxifloxacin was done at 296 nm. Method was found to be selective, linear (r = 0.9997), accurate (recovery, 97.30–99.99%) and precise (RSD, ≤1.72%) in the selected concentration range of 10–1000 ng/ml. Detection and quantitation limit of moxifloxacin in aqueous humor were 0.75 and 2.5 ng/ml, respectively. The aqueous humor levels of moxifloxacin after single topical instillation in three formulations, i.e. moxifloxacin solution (Moxi-SOL), anionic nanoparticles (Moxi-ANP) and cationic nanoparticles (Moxi-CNP) were investigated. A fourfold increase in the relative bioavailability was observed with the Moxi-CNP (AUC_0→t, 3.14 μg h/ml) compared with Moxi-SOL (AUC_0→t, 0.79 μg h/ml) and Moxi-ANP (AUC_0→t, 0.72 μg h/ml) formulation. The results indicate that the cationic nanoparticle increases ocular bioavailability of moxifloxacin and prolong its residence time in the eye.     

Development of a validated liquid chromatographic method for determination of related substances of telmisartan in bulk drugs and formulations

A simple and rapid reversed phase liquid chromatographic method for separation and determination of the related substances of telmisartan (TLM) was developed and validated. The chromatographic separation was achieved on Lichrospher RP-18 column (250 × 4.6 mm, 5 μm), using 20 mM ammonium acetate containing 0.1% (v/v) triethylamine (pH adjusted to 3.0 with trifluoroacetic acid) and acetonitrile as mobile phase at 25 °C. The detection was performed at 254 nm. The method was validated and found to be robust, precise, specific and linear between 0.37 and 500 μg/mL. The limits of detection and quantification of telmisartan were 0.11 and 0.37 μg/mL, respectively. The method was successfully applied to quantify related substances and assay of TLM in bulk drugs and commercial tablets. The related substances relate to a novel synthetic route and different from those A-H impurities reported by European Pharmacopeia.     

Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile–0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1–50 μg/ml (R² = 0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17 ± 0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 μg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release.     

Development and validation of an enantioselective and chemoselective HPLC method using a Chiralpak IA column to simultaneously quantify (R)-(+)- and (S)-(−)-lansoprazole enantiomers and related impurities

An accurate and reproducible high-performance liquid chromatographic (HPLC) method has been developed and validated for the direct separation of individual enantiomers of lansoprazole, a potent proton pump inhibitor belonging to the family of the substituted benzimidazoles. The enantiomers were resolved on a Chiralpak IA by using a mobile phase consisting of methyl-tert-butyl ether (MtBE)–ethyl acetate (EA)–ethanol (EtOH)–diethylamine (DEA) in the ratio 60:40:5:0.1 (v/v/v/v). Baseline separation of the enantiomers of lansoprazole was obtained with a resolution factor of 8.14. The standard curves for the two enantiomers were linear (r² > 0.999) in the concentration range of 10–80 μg/ml with a working concentration of about 60 μg/ml for each enantiomer. Apparent recovery was 100.8% with a relative standard deviation less than 2%. The limit of quantization for each enantiomer of lansoprazole was 0.22 μg/ml. The intra-day precisions were in the range of 0.21–0.36 and 0.59–0.66 while the inter-day precisions were in the range of 0.55–1.24 and 0.66–1.19% in terms of retention times and area response RSD% for (R)-(+)- and (S)-(−)-lansoprazole, respectively. The method was also able to resolve impurities from the enantiomers of lansoprazole.     

Development and validation of an HPLC-DAD method for bis(12)-hupyridone and its application to a pharmacokinetic study

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C₁₈ 5 μm column (length, 250 mm; inner diameter, 4.6 mm) using a mixture of water–acetonitrile–trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1 mL/min, with detection at 229 nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1–7.5 μg/mL with r² = 0.9991. The limit of detection and quantification of this method were 0.05 μg/mL and 0.1 μg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5 mg/kg and intraperitoneal administration of 10 and 20 mg/kg in rats.     

Simultaneous determination of Maillard reaction impurities in memantine tablets using HPLC with charged aerosol detector

A gradient high performance liquid chromatographic (HPLC) method using charged aerosol detection (CAD) was developed for the simultaneous determination of impurities formed by the Maillard reaction in memantine tablets. These impurities were a memantine-lactose adduct (ML), a memantine-dimethylamino glycine adduct (DMAG), a memantine-galactose adduct (MGAL), and a memantine-glucose adduct (MGLU). The chromatographic separation was performed on a Synergy Hydro RP column (100 mm × 3 mm, 2.5 μm particles) from Phenomenex with gradient elution using mobile phases consisting of 0.6% (v/v) of heptafluorobuturic acid (HFBA) in two acetonitrile-isopropyl alcohol-water mixtures. The method validation for the impurities included linearity, accuracy by spike recovery, precision, limits of detection and quantitation, and robustness. The method was sensitive for these non-chromophoric impurities down to 0.4-0.6 μg/mL (0.02-0.03% of the memantine drug substance). The effect of mobile phase HFBA concentration on chromatographic resolution and peak shape was investigated, and the effect of sample diluent acidification on method accuracy via spike recovery was studied. The operational simplicity of the CAD detector for routine quality control has been demonstrated.     

LC and LC–MS/MS study of forced decomposition behavior of anastrozole and establishment of validated stability-indicating analytical method for impurities estimation in low dose anastrozole tablets

Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. Separation of anastrozole from its potential impurities, degradation products and five anastrozole related compounds as main impurities were achieved on Inertsil ODS-3V, 250 mm × 4.6 mm i.d, 5 μm analytical column using reversed phase high performance liquid chromatography (RP-HPLC). The elution of impurities employed time dependent gradient programmed mobile phase consisting of water as mobile phase-A and acetonitrile as mobile phase-B at column flow rates of 1 ml/min and at 215 nm UV detection. The same method was also extended to LC–MS/MS studies which were carried out to identify the degradation product. The method developed was established to have sufficient intermediate precision as similar separation was achieved on another instrument handled by a different operator. The LOQ for anastrozole related compound-A (RC-A), related compound-B (RC-B), related compound-C (RC-C), related compound-D (RC-D), related compound-E (RC-E) and anastrozole were 0.05, 0.03, 0.03, 0.06, 0.06 and 0.06 μg ml⁻¹ respectively. The linearity of the proposed method for all the above related compounds was investigated in the range of LOQ to 0.600 μg ml⁻¹ respectively. The specificity was established through peak purity testing using a photo-diode array detector. Method was validated according to ICH guidelines and statistical analysis of the data proved to be suitable for stability testing at quality control.     

Development and validation of a high-performance liquid chromatographic method for bioanalytical application with rimonabant

A simple and feasible high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of rimonabant in human plasma. The chromatographic separation was carried out in a Hypersil BDS, C₁₈ column (250 mm × 4.6 mm; 5 μm). The mobile phase was a mixture of 10 mM phosphate buffer and acetonitrile (30:70, v/v) at a flow rate of 1.0 ml/min. The UV detection was set at 220 nm. The extraction recovery of rimonabant in plasma at three quality control (QC) samples was ranged from 84.17% to 92.64%. The calibration curve was linear for the concentration range of 20–400 ng/ml with the correlation coefficient (r²) above 0.9921. The method was specific and sensitive with the limit of quantification of 20 ng/ml. The accuracy and precision values obtained from six different sets of QC samples analyzed in separate occasions ranged from 88.13% to 106.48% and 0.13% to 3.61%, respectively. In stability tests, rimonabant in human plasma was stable during storage and assay procedure. The method is very simple, sensitive and economical and the assay was applied to human plasma samples in a clinical (pharmacokinetic) study of rimonabant.     

Development of a high performance liquid chromatography method for quantification of PAC-1 in rat plasma

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm particle size, Zhonghuida) protected by a ODS guard column (10 mm × 4.6 mm i.d., 5 μm particle size), using acetonitrile–methanol–phosphate buffer (pH 3.0, 30 mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0 mL/min, and wavelength of the UV detector was set at 281 nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05–20 μg/mL (r > 0.99). The lower limit of quantification was evaluated to be 50 ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.     

High-throughput determination of faropenem in human plasma and urine by on-line solid-phase extraction coupled to high-performance liquid chromatography with UV detection and its application to the pharmacokinetic study

An automated system using on-line solid-phase extraction and HPLC with UV detection was developed for the determination of faropenem in human plasma and urine. Analytical process was performed isocratically with two reversed-phase columns connected by a switching valve. After simple pretreatment for plasma and urine with acetonitrile, a volume of 100 μl upper layer of the plasma or urine samples was injected for on-line SPE column switching HPLC-UV analysis. The analytes were retained on the self-made trap column (Lichrospher C₁₈, 4.6 mm × 37 mm, 25 μm) with the loading solvent (20 mM NaH₂PO₄ adjusted pH 3.5) at flow rate of 2 ml min⁻¹, and most matrix materials were removed from the column to waste. After 0.5 min washing, the valve was switched to another position so that the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (acetonitrile–20 mM NaH₂PO₄ adjusted pH 3.5, 16:84, v/v) at flow rate of 1.5 ml min⁻¹, and then separated on the analytical column (Ultimate™ XB-C₁₈, 4.6 mm × 50 mm, 5 μm).The complete cycle of the on-line SPE preconcentration purification and HPLC separation of the analytes was 5 min. Calibration curves with good linearities (r = 0.9994 for plasma sample and r = 0.9988 for urine sample) were obtained in the range 0.02–5 μg ml⁻¹ in plasma and 0.05–10 μg ml⁻¹ in urine for faropenem. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully utilized to quantify faropenem in human plasma and urine to support the clinical pharmacokinetic studies.     

Rapid and sensitive assay for trantinterol,a novel β2-adrenoceptor agonist,in human plasma using liquid chromatography–tandem mass spectrometry

A rapid and sensitive assay for trantinterol, a novel β₂-adrenoceptor agonist, in human plasma has been developed. Samples containing the analyte and internal standard, clenbuterol, were analyzed by liquid chromatography–tandem mass spectrometry after liquid–liquid extraction with diethyl ether:dichloromethane (60:40, v/v). Separation was performed on a Venusil MP C₁₈ column (50 mm × 4.6 mm, 5 μm) using methanol:1% formic acid (50:50, v/v) as mobile phase and monitored by multiple reaction monitoring of the precursor-to-product ion transitions of trantinterol at m/z 311.2 → 238.1 and clenbuterol at m/z 277.2 → 203.1. The total run time was only 1.5 min and the method was linear over the concentration range 1–1000 pg/mL with a lower limit of quantitation of 1 pg/mL. Intra- and inter-day precisions (relative standard deviation) were below 7% and 12%, respectively, with accuracy (relative error) below 8%. The method was successfully applied to a pharmacokinetic study involving oral administration of a 50 μg trantinterol tablet to healthy volunteers.     

Determination of SNX-2112, a selective Hsp90 inhibitor,in plasma samples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A sensitive and specific reversed-phase high-performance liquid chromatography method with ultraviolet detection has been developed and validated for the identification and quantification of SNX-2112 in rat plasma. Following sample preparation using liquid–liquid extraction, the analytes were separated by the mobile phase acetonitrile–water (40:60, v/v) with an Agilent RP-HPLC column (ZORBAX SB-C18, 5 μm, 4.6 mm × 250 mm) at a flow rate of 1 ml/min, column temperature of 30 °C and detection wavelength of 251 nm. The retention time of SNX-2112 was 11.2 min. A good linear relationship was obtained in the concentration range studied (0.07–21 μg/ml, R² > 0.9982), and the LLOD and LLOQ for SNX-2112 were 0.02 and 0.07 μg/ml, respectively. The mean absolute recovery of SNX-2112 in plasma ranged from 88.58 to 99.61% at the studied concentrations. The intra- and inter-batch relative standard deviations were 1.7–3.5 and 1.9–4.4%, respectively. This method was successfully applied to pharmacokinetic studies in rats after intravenous administration of SNX-2112.     

Developing and optimizing a validated isocratic reversed-phase high-performance liquid chromatography separation of nimodipine and impurities in tablets using experimental design methodology

In the present study an isocratic reversed-phase high-performance liquid chromatography was investigated for the separation of nimodipine and impurities (A, B and C) using statistical experimental design. Initially, a full factorial design was used in order to screen five independent factors: type of the organic modifier – methanol or acetonitrile – and concentration, column temperature, mobile phase flow rate and pH. Except pH, the rest examined factors were identified as significant, using ANOVA analysis. The optimum conditions of separation (optimum values of significant factors) determined with the aid of central composite design were: (1) mobile phase: acetonitrile/H₂O (67.5/32.5, v/v), (2) column temperature 40 °C and (3) mobile phase flow rate 0.9 ml/min. The proposed method showed good prediction ability (observed–predicted correlation). The analysis was found to be linear, specific, precise, sensitive and accurate. The method was also studied for robustness and intermediate precision using experimental design methodology. Three commercially available nimodipine tablets were analyzed showing good % recovery and %RSD. No traceable amounts of impurities were found in all products.     

PK and tissue distribution of docetaxel in rabbits after i.v. administration of liposomal and injectable formulations

A simple and sensitive HPLC method was established and validated for the determination of docetaxel (DTX) in rabbit plasma and tissue samples. Biosamples were spiked with paclitaxel as an internal standard and pre-treated by solid phase extraction (SPE). Sample separation was performed on a reverse-phase HPLC column at 30 °C by using a mobile phase of acetonitrile–methanol–0.02 M ammonium acetate buffer (pH 5.0) (20:47.5:32.5, v/v/v) at flow rate of 1.0 mL/min The UV absorbance of the samples was measured at the wavelength of 230 nm. The standard curves were linear over the ranges of 0.02525–2.525 μg/mL for plasma, 1.010–202.00 μg/g for lung, 0.202–20.20 μg/g for spleen, liver and kidney, 0.202–10.10 μg/g for heart and stomach, 0.0505–2.02 μg/g for brain, respectively. The limits of quantification (LOQ) were 10.0 ng/mL in the plasma samples and 20.0 ng/g in the tissue samples, respectively. The analysis method was successfully applied to pharmacokinetics and tissue distribution studies of DTX liposomes and DTX injection after i.v. administration to the rabbits. The results showed that the liposome carrier led to a significant difference in pharmacokinetics and tissue distribution profile compared to the conventional DTX injection.     

Development and validation of a liquid chromatographic method for the simultaneous determination of aniracetam and its related substances in the bulk drug and a tablet formulation

Simultaneous determination of aniracetam and its related impurities (2-pyrrolidinone, p-anisic acid, 4-p-anisamidobutyric acid and (p-anisoyl)-4-methyl-2-pyrrolidinone) was accomplished in the bulk drug and in a tablet formulation using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS-CN column (150 mm × 4.0 mm, 5 μm) using a gradient elution program with solvent A composed of phosphate buffer (pH 4.0; 0.010 M) and solvent B of acetonitrile-phosphate buffer (pH 4.0; 0.010 M) (90:10, v/v). The flow rate of the mobile phase was 1.0 mL min⁻¹ and the total elution time, including the column re-equilibration, was approximately 20 min. The UV detection wavelength was varied appropriately among 210, 250 and 280 nm. Injection volume was 20 μL and experiments were conducted at ambient temperature. The developed method was validated in terms of system suitability, selectivity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities, short term and long term stability of the analytes in the prepared solutions and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of aniracetam and its studied related impurities.     

LC–MS/MS studies of ritonavir and its forced degradation products

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra^® C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.     

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 1 mg/ml analyte test solution is chromatographed.     

Liquid chromatographic determination of lumiracoxib in pharmaceutical formulations

A stability-indicating reversed-phase liquid chromatography (LC) method was developed and validated for the determination of lumiracoxib in pharmaceutical formulations. The LC method was carried out on a Synergi fusion C₁₈ column (150 mm × 4.6 mm), maintained at 30 °C. The mobile phase was composed of phosphoric acid (25 mM; pH 3.0)/acetonitrile (40:60, v/v), run at a flow rate of 1.0 mL/min, and detection at 272 nm. The chromatographic separation was obtained within 10 min and it was linear in the concentration range of 10–100 μg/mL (r² = 0.9999). Validation parameters such as the specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range. Stress studies were carried out and no interference of the degradation products was detected. Moreover, the proposed method was successfully applied for the assay of lumiracoxib in pharmaceutical formulations.     

Quantitation of the tacrine analogue octahydroaminoacridine in human plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of octahydroaminoacridine in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5 μm, 100 mm × 4.6 mm) using acetonitrile:10 mM ammonium acetate:formic acid (30:70:1, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1 → 175.1 and of the I.S. at m/z 264.1 → 58.0. The method was linear in the range 0.01–10 ng/ml with a lower limit of quantitation of 0.01 ng/ml. Intra- and inter-day precisions measured as relative standard deviation were <3.15% and <5.01%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of a tablet containing 4 mg octahydroaminoacridine succinate to healthy volunteers. Pharmacokinetic parameters for octahydroaminoacridine include C_max 1.19 ± 0.53 ng/ml, T_max 0.77 ± 0.17 h, AUC_0−t 3.42 ± 1.01 ng h/ml and t_1/2 2.89 ± 0.56 h.     

Simultaneous HPLC analysis of pseudophedrine hydrochloride,codeine phosphate,and triprolidine hydrochloride in liquid dosage forms

An HPLC method using UV detection is proposed for the simultaneous determination of pseudophedrine hydrochloride, codeine phosphate, and triprolidine hydrochloride in liquid formulation. C18 column (250 mm × 4.0 mm) is used as the stationary phase with a mixture of methanol:acetate buffer:acetonitrile (85:5:10, v/v) as the mobile phase. The factors affecting column separation of the analytes were studied. The calibration graphs exhibited a linear concentration range of 0.06–1.0 mg/ml for pseudophedrine hydrochloride, 0.02–1.0 mg/ml for codeine phosphate, and 0.0025–1.0 mg/ml for triprolidine hydrochloride for a sample size of 5 μl with correlation coefficients of better than 0.999 for all active ingredients studied. The results demonstrate that this method is reliable, reproducible and suitable for routine use with analysis time of less than 4 min.