Simultaneous Determination of Pioglitazone and Glimepiride by High-Performance Liquid Chromatography

A rapid and accurate HPLC method has been developed for simultaneous determination of pioglitazone and glimepiride. Chromatographic separation of the two pharmaceuticals was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm, 5 m) with a 45:35:20 (v/v) mixture of 0.01 m triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid), acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL min^–1, and detection at 228 nm. Separation was complete in less than 10 min. The method was validated for linearity, accuracy, precision, limit of quantitation, and robustness [1, 2]. Linearity, accuracy, and precision were found to be acceptable over the ranges 2.50–30.00 g mL^–1 for pioglitazone and 0.10–10.00 g mL^–1 for glimepiride.     

LC with Column-Switching and Spectrophotometric Detection for Determination of Risperidone and 9-Hydroxyrisperidone in Human Serum

An automated high performance liquid chromatography with column-switching and ultraviolet detection was developed for the analysis of risperidone and 9-hydroxyrisperidone. The method needs minimum sample preparation and is useful for the detection down to a limit of 1 ng mL⁻¹. Sample clean-up of serum was carried out on a CN 20 μm SPE-column using 8% (v/v) acetonitrile in water. Chromatographic separation was performed on ODS Hypersil C18 material with 38% (v/v) acetonitrile and 0.4% (v/v) TEMED in water. Application of the method to the analysis of serum samples confirmed its suitability for therapeutic drug monitoring of risperidone and 9-hydroxyrisperidone.     

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5?×?4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min^?1 and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100?×?4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5 % triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min^?1 and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL^?1.

LC Analysis of JS38, a Novel Antineoplastic Medicine, in the Plasma, Urine, Bile, Feces, and Other Tissues of Rats

An RP-LC method has been developed for analysis of JS38 in the plasma, urine, bile, feces, and important tissues of rats. Chromatography was performed on a C₁₈ analytical column with 80:20 (%, v/v) acetonitrile–phosphate buffer (0.5% phosphoric acid and 0.3% TEA adjusted to pH 5.0) as mobile phase at a flow-rate of 1.0 mL min^?1. Eluted compounds were detected at 400 nm by ultraviolet diode-array detection (DAD). The method was validated for linearity, accuracy (recovery from biological matrixes such as plasma, urine, bile, feces, and important organs), repeatability (within-day and between-day precision), and stability. The results indicate that the method is suitable for pre-clinical pharmacokinetic study of JS38.     

Focused Microwave-Assisted Extraction and LC Determination of Ketoprofen in the Presence of Preservatives in a Pharmaceutical Cream Formulation

A simple and rapid open-vessel focused microwave-assisted extraction (FMAE) method followed by LC analysis was developed for the determination of ketoprofen lysine salt in the presence of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate preservatives in topical cream. Extraction were performed in acetone/potassium dihydrogenphosphate (25 mM, pH 3.0) (70:30 v/v) by reaching a target temperature of 65 °C in a 10 min linear ramp. The chromatographic separation was performed on a Discovery RP-Amide C16 column (250 × 4.6 mm I.D., 5 μm particle size). The optimal mobile phase consisted of acetonitrile/potassium dihydrogen phosphate 25 mM adjusted to pH 3.0 with phosphoric acid (50:50 v/v). The complete analytical procedure was validated with regard to limit of quantification, linearity, precision and accuracy. The method was linear over the concentration range of 0.08–0.12 mg mL⁻¹; the relative standard deviations of intra- and inter-day assays were 1.9–2.3 and 1.8% respectively. The limit of quantification was 0.54 μg mL⁻¹. The proposed method shows many advantages as short extraction time, little solvent consumption without requiring further sample clean-up steps before liquid chromatographic analysis and is proposed for vast scale screening of cream dosage forms aimed to the detection of counterfeit and substandard drugs.

     

High-Performance Liquid Chromatographic Determination of Cinnarizine in Workplace Air

Summary Cinnarizine is a pharmaceutical drug used in the treatment of cerebral and peripheral vascular diseases. A reversed-phase liquid chromatographic method with fluorescence detection has been developed for determination of the drug in workplace air. Air sampling in the workplace is performed on perchlorovinyl filters (FPP), the filters are extracted with methanol for 40 min, and the extract (50 L) is injected and separated on a 250 mm × 4.6 mm i.d., 5 m particle, C₈ reversed-phase column with 1% ammonium acetate (pH 4.5)–acetonitrile, 1:4 (v/v), as mobile phase at a flow rate of 1 mL min^–1.     

Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min⁻¹. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL⁻¹ (r = 0.9999) for TR and 40–160 μg mL⁻¹ (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.

     

Rapid analysis of pyrethroids in whole urine by high-performance liquid chromatography using a monolithic column and off-line preconcentration in a restricted access material cartridge

A rapid high-performance liquid chromatography (HPLC) method using a monolithic column with UV detection at 238 nm was developed for the determination of fenpropathrin, betacyfluthrin, deltamethrin, and permethrin (cis and trans isomers) in whole urine. The method is based on the use of a monolithic chromatographic column and a restricted access material (RAM) cartridge for sample preparation. The mobile phase was water/acetonitrile (42:58 v/v), the flow rate was 3 mL min^–1, and chromatographic separation was carried out in 10 min. The separation of cis and trans isomers of permethrin was also possible under the above-mentioned conditions. Detection limits in reconstituted whole urine samples were between 0.9 g L^–1 for betacyfluthrin and 4.4 g L^–1 for fenpropathrin and trans-permethrin. Recoveries for urine samples spiked with different amounts of pyrethroids (between 19 g L^–1 and 75 g L^–1) were in the 70±6 to 90±7% range.     

An Improved Narrow-Bore LC Method for Quantification of Alfuzosin in Pharmaceutical Formulations

A simple rapid and stability-indicating LC method using a narrow-bore column has been developed, fully validated, and applied to the quantification of alfuzosin in pharmaceutical formulations. Chromatography was achieved isocratically on a narrow-bore, 5-μm particle size, C₈ analytical column. The mobile phase was a 35:65 (v/v) 0.0125 m ammonium formate–acetonitrile at a flow rate of 0.35 mL min^?1. Detection was by UV absorption at 245 nm. Evaluation over the range 200–800 ng mL^?1 revealed linearity was good. Limits of detection and quantification for alfuzosin were 22.9 and 69.5 ng mL^?1, respectively. Intra-day and inter-day RSD were less than 6.4%, and the relative percentage error was less than ?1.7% (n = 5). Accelerated degradation performed under different stress conditions including oxidation, hydrolysis, and heat, proved the selectivity of the procedure. The method was successfully used for quality-control and content-uniformity testing of commercial tablets.     

Determination of Benzimidazole Fungicides by HPLC with Fluorescence Detection After Micellar Extraction

An analytical method was developed to determine the benzimidazole fungicides and their residues (benomyl, carbendazim, thiabendazole and fuberidazole) in real water samples. Analyses were performed by reverse phase (RP) HPLC with direct fluorescence detection with mobile phase methanol:water, 40:60 (v/v) with 0.6% (v/v) ammonia. The extraction of analytes from water samples was performed with the use of micellar systems. Specifically, oligoethylene glycol monoalkyl ether (Genapol X-080) and polyoxyethylene 10 lauryl ether (POLE) were used as extractants. The recoveries of fungicides obtained in spiked water samples ranged from 68% to 94% for Genapol and from 68% to 96% for POLE. The limit of detection (LOD) was lower than 6 g L^–1 for carbendazim, 7 g L^–1 benomyl, 0.15 g L^–1 for thiabendazole and 0.01 g L^–1 for fuberidazole in both surfactants.     

Determination of the Enantiomers of Tamsulosin Hydrochloride and its Synthetic Intermediates by Chiral Liquid Chromatography

A chiral liquid chromatographic method is described for the determination of the enantiomers of tamsulosin hydrochloride and its synthetic intermediates. Enantioseparation was achieved on a Chiralcel OD-R column (250 mm × 4.6 mm, 10 m) using a mobile phase consisting of a mixture of 0.5 mol L^–1 sodium perchlorate and acetonitrile (80:20, v/v, pH 4.0). The flow rate was 0.4 mL min^–1 and detection was at 223 nm. Excellent enantiomer separations were achieved for tamsulosin hydrochloride and its synthetic intermediates. No other methods are available for the separation of these enantiomers. The method developed in this study has been successfully applied for purity control.     

LC-DAD and LC–MS–MS Analysis of Piperidine Alkaloids of Lobelia inflata L. (In Vitro and In Vivo)

An LC-DAD method was developed for determination of lobeline from in vitro and in vivo cultures of Lobelia inflata. Samples were extracted with 0.1 N HCl–acetonitrile (1:1, v/v), and purified by solid-phase extraction. Optimized conditions resulted in high recovery. LC separations were performed on an Eurosphere C8 reversed-phase column using 30:70 (v/v) acetonitrile–0.1% trifluoroacetic acid as a mobile phase. Quantitative determination of lobeline was performed by external standard method at 250 nm, in the range of 2.4–80 μg mL⁻¹. Validation studies proved that the repeatability of the method was good and the recovery was satisfactory. In vitro organized cultures contained considerable amount of lobeline (herb: 175 μg g⁻¹, root: 100 μg g⁻¹). When these cultures were transplanted into the open field, the lobeline content increased significantly (herb: 323 μg g⁻¹, root: 833 μg g⁻¹). Plants obtained from seed propagation contained 382 μg g⁻¹ lobeline in the herb. For direct characterization of di-substituted piperidine alkaloids in extracts of L. inflata, tandem mass spectrometric method was developed using electrospray ionization. Analysis was performed in the positive ion mode on a triple quadropole LC–MS system. LC separations were achieved on Eurosphere C8 column with a modified mobile phase (acetonitrile–30 mM ammonium formate, pH 2.80) to ensure proper molecular ionization. The identification and structural elucidation of the alkaloids were performed by comparing their changes in molecular mass (ΔM), full-scan MS–MS spectra with those of lobeline, norlobelanine and lobelanidine. These alkaloids and ten other derivatives were identified in the plant extracts. Three piperidine alkaloids were reported in L. inflata for the first time.

     

Retention Behavior of Anti-Arrhythmic Drugs on the Chromolith Performance RP 18 Column. Liquid Chromatographic Determination of Mexiletine Hydrochloride

The retention behavior in liquid chromatography of a series of anti-arrhythmic drugs is described. Chromatographic analysis was performed on a Chromolith Performance ODS column with acetonitrile–phosphate buffer mixtures as mobile phases. The effects of the proportion of organic solvent (from 20 to 90%), phosphate buffer pH (from 2.73 to 7.5), flow rate (from 1 to 6 mL min^–1), and isocratic or gradient elution on the retention of the compounds was studied. Mexiletine hydrochloride was determined in the pure substance and in capsules by isocratic liquid chromatography with 20:80 (v/v) acetonitrile–0.007 M phosphate buffer, pH 3.0, as mobile phase at 2 mL min^–1. Methanol was found to be a suitable solvent for extraction of the active substance from capsules. The calibration plot was linear (r=0.9999) in the concentration range 1.0 to 6.0 g mL^–1. The proposed method is selective, precise (RSD=0.37%), and accurate (recovery=100.08%).Revised: 28 January and 2 March 2004     

Liquid chromatographic–electrospray mass spectrometric determination of cyclosporin A in human plasma

A rapid, sensitive and selective liquid chromatography–mass spectrometry (LC–MS) assay has been developed for determination of cyclosporin A (CyA) in human plasma; cyclosporin B (CyB) was used as internal standard (IS). The method utilized a combination of a column-switching valve and a reversed-phase symmetry column. The mobile phase was a 25:75 (v/v) mixture of 10% aqueous glacial acetic acid and acetonitrile. Running time per single run was less than 10 min. Sample preparation included C₈ SPE of human plasma spiked with the analyte and internal standard, evaporation of the eluate to dryness at 50°C under N₂ gas, and finally reconstitution in the mobile phase. Detection of cyclosporin A and the IS was performed in selected ion-monitoring mode at m/z 601.3 and 594.4 Da for CyA and IS, respectively. Quantitation was achieved by use of the regression equation of relative peak area of cyclosporin to IS against concentration of cyclosporin. The method was validated according to FDA guideline requirements. The linearity of the assay in the range 5.0–400.0 ng mL^–1 was verified as characterized by the least-squares regression line Y=(0.00268±1.9×10^–4)X+(0.00078±1.8×10^–3), correlation coefficient, r=0.9986±1.1×10^–3 (n=48). Intra and inter-day quality-control measurements in the range 5.0–350.0 ng mL^–1 revealed almost 100% accuracy and 9% CV for precision. The mean absolute recovery of CyA was found to be 84.01±9.9% and the respective relative recovery was 100.3±9.19. The limit of quantitation (LOQ) achieved was 5 ng mL^–1. Eventually, stability testing of the analyte and IS in plasma or stock solution revealed that both chemicals were very stable when stored for long or short periods of time at room temperature or –20°C.     

Determination of Lercanidipine Hydrochloride and Its Impurities in Tablets

The reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for determination of lercanidipine hydrochloride and its synthetic impurities, degradation and oxidative products in Carmen^® tablets. The best separation was performed on Zorbax SB C18 column, 250 x 4.6 mm, particle size 5 m. Acetonitrile-water-triethylamine 55:44.8:0.2 (v/v/v) was used as a mobile phase with flow rate 1 mL min^–1. pH was adjusted to 3.0 with orthophosphoric acid. UV detection was performed at 240 nm. Duration of chromatographic run was about 12 min for six examined compounds. The chromatographic conditions for the determination of lercanidipine hydrochloride and its related substances were the same, but the concentration of lercanidipine hydrochloride was 0.03 mg mL^–1 for assay and 0.3 mg mL^–1 for related substances. The validation of the method performance characteristics (figures of merits, quality of parameters) was established to be adequate for the intended use. The evaluation of number of parameters, such as selectivity, linearity, accuracy, specificity, precision (repetability and reproducibility), sensitivity and detection and determination limits is entailed.Acknowledgements This work was supported by the Institute of Pharmacy of Serbia, Belgrade and by the Ministry for Science, Technology and Development of Serbia, Contact: 1458     

LC−UV Analysis of N-{3-(2,4-Dioxo-1,4-dihydro-2H-quinazolin-3-yl)propyl}-N-[4-{3-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)propyl-amino}butyl]acetamide (SP-8203) in Rat Plasma,Urine, and Gastrointestinal Tract Samples

A simple, rapid, and reproducible reversed-phase LC method with UV detection at 215 nm has been developed for analysis of SP-8203 in rat samples. A C₁₈ column was used with 3,000:1,050 (v/v) 0.01 m K₂HPO₄ buffer (pH 3)–acetonitrile as mobile phase at a flow rate of 1.7 mL min⁻¹ at 50 °C. Samples were extracted with dichloromethane containing ondansetron (internal standard). Detection limits for SP-8203 in plasma, urine, and gastrointestinal tract samples were 0.05, 0.5, and 10 μg mL⁻¹, respectively. The method was suitable for pharmacokinetic study of SP-8203 in rats after intravenous administration.

     

Quantitative Determination of Carisoprodol and its Metabolites in Equine Urine and Serum by Liquid Chromatography-Tandem Mass Spectrometry

Cefuroxime is a broad-spectrum second-generation bactericidal cephalosporin antibiotic active against β-lactamase-producing strains. Anti-cefuroxime, the geometric isomer of cefuroxime, might be present in cefuroxime dosage forms as a process-related impurity and possible degradation product. In the work discussed in this paper a precise and sensitive micellar liquid chromatographic (MLC) method for stability testing of cefuroxime axetil and anti-cefuroxime axetil in tablets, using benzoic acid as internal standard, was developed and validated. MLC was performed on an XTerra C₁₈ reversed-phase column at 50 °C with 8:92 (v/v) acetonitrile–20 mM sodium dodecyl sulphate, pH 2.5, as mobile phase at a flow rate of 1.5 mL min^?1. Detection was at 280 nm. Under these conditions the retention time and retention factor were of 6.65 min and 4.57, respectively, for cefuroxime axetil and 11.45 min and 8.59, respectively, for anti-cefuroxime axetil, indicating that the compounds were well separated. RSD values for quantification of cefuroxime axetil and anti-cefuroxime axetil were 0.39 and 1.7%, respectively, indicating the precision of the MLC method was good. The method is sensitive—LOD=0.5 μg mL^?1 and LOQ=1.5 μg mL^?1 for anti-cefuroxime axetil—and reproducible, with good recovery values.     

HPLC Determination and Pharmacokinetic Study of Valdecoxib in Human Plasma

A sensitive, simple, and accurate high-performance liquid chromatographic method has been developed for determination of valdecoxib and the internal standard rofecoxib in human plasma. Protein was precipitated from plasma samples by addition of perchloric acid (HClO₄); the drug was then extracted with diethyl ether. Separation was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm i.d., 5 m particles) with ammonium acetate buffer-acetonitrile, 60:40 (v/v), containing 0.1% TEA, pH 6.5, as mobile phase. Detection and quantification were performed by UV-visible detection at 239 nm. Detection and quantification limits were 3 and 5 ng mL^–1, respectively. The linear concentration range for valdecoxib was 5–400 ng mL^–1. The validated RP HPLC method was used for determination of the pharmacokinetic data for the drug in humans.     

Application of solid-phase extraction for determination of phenolic compounds in barrique wines

A fast, selective and sensitive chromatographic method has been developed for determination of gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, benzoic, ferulic, sinapic, cinnamic, and ellagic acids and p-hydroxybenzaldehyde, vanillin, syringaldehyde, 2-furfural, 5-methylfurfural, and 5-methoxyfurfural. The compounds from untreated wine samples were pre-concentrated and cleaned using solid-phase extraction on RP-105 polymeric sorbent. The cartridge was conditioned with methanol and water. Co-extracted ballast substances were rinsed from the sorbent with 0.1 mol L^–1 hydrochloric acid–methanol, 1:4 (v/v). Retained phenolic compounds were selectively eluted with diethyl ether. A linear mobile phase gradient containing 0.3% acetic acid and methanol was used for final baseline chromatographic separation on a Hypersil BDS C18 column. Limits of detection (LOD=3s_bl) in the range 5.2 to 181.2 g L^–1, resolution (R) better than 1.7, and repeatability of 2.7–5.1% (RSD for real samples) were achieved. The method was applied for quantification of individual phenolic compounds in barrique wines.     

Rapid RP-LC Method with Fluorescence Detection for Analysis of Fexofenadine in Human Plasma

A rapid and sensitive reversed-phase high-performance liquid chromatographic method for analysis of fexofenadine in human plasma has been developed and optimized. The analytes were extracted from biological samples by solid-phase extraction on hydrophilic–lipophilic balance cartridges. LC separation was performed on a C₁₈ analytical column (125 mm × 4 mm i.d., 5-μm particles) with 42:58 (v/v) acetonitrile–water adjusted to pH 2.7 with 85% orthophosphoric acid as mobile phase. Fluorescence detection was performed with excitation at 230 nm and emission at 290 nm. The total time for chromatographic separation was 7 min. The method was validated in accordance with EU guidelines by analysis of plasma samples fortified with fexofenadine at concentrations between 0.05 and 800 ng mL⁻¹. Calibration plots were linear in this range. Mean recovery was typically 94.03% and the detection limit was 0.05 ng mL⁻¹. The time required for quantitative analysis is shorter than that required by other methods.