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.     

Determination of Nisoldipine and Its Impurities in Pharmaceuticals

A method has been established for separation of nisoldipine and impurities, for example reactants, products of side-reactions, and photodegradation products, by HPTLC on LiChrospher Si 60 F_254s plates with detection at 280 nm. The mobile phase, cyclohexane–ethyl acetate–toluene, 7.5:7.5:10 (v/v), enables acceptable resolution of nisoldipine, in large excess, and possible impurities. Regression coefficients (r 0.997), recovery (98–108%), and determination limit (0.02–0.2%) were validated and found to be satisfactory. The method is convenient for quantitative analysis and purity control of nisoldipine in its raw material and dosage forms.     

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.     

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     

A Stability-Indicating LC Method for Assay of Topotecan Hydrochloride

This paper describes the development of a stability-indicating high-performance liquid chromatographic (HPLC) method for quantitative determination of topotecan hydrochloride, a semi-synthetic derivative of camptothecin and anti-tumor drug with topoisomerase I-inhibitory activity. Chromatographic separation was achieved on a C₁₈ column with a mixture of phosphate buffer and acetonitrile as mobile phase. The method was validated for linearity, accuracy, precision, and robustness. Forced degradation studies were performed by treating bulk samples of topotecan hydrochloride with acid (0.5 M hydrochloric acid), base (0.5 M sodium hydroxide), oxidizing agent (10% v/v hydrogen peroxide), heat (60 °C), and UV light (254 nm).     

High-performance liquid chromatographic determination of diltiazem in human plasma after solid-phase and liquid–liquid extraction

A selective, sensitive, and accurate high-performance liquid chromatographic method for determination of diltiazem in plasma samples has been developed and validated. The effects of mobile phase composition, buffer concentration, mobile phase pH, and concentration of organic modifiers on retention of diltiazem and internal standard were investigated. Solid-phase and liquid–liquid extraction were examined and proposed for isolation of the drug and elimination of endogenous plasma interferences. A 5 m Lichrocart Lichrospher 60 RP-select B chromatographic column was used; the mobile phase was acetonitrile–0.025 mol L^–1 KH₂PO₄ (pH 5.5), 35:65 ( v / v) at a flow-rate of 1.5 mL min^–1. The detection wavelength was 215 nm. The calibration plots were linear in the concentration range 20.0–500.0 ng mL^–1. The method has been implemented to monitor diltiazem levels in patient samples.     

Optimization and Validation of an HPLC-UV Method for Determination of Tranexamic Acid in a Dosage Form and in Human Urine

A simple and reliable high-performance liquid chromatographic method with UV detection at 245 nm has been developed and validated for determination of tranexamic acid (TA) in a dosage form and in human urine. Before injection samples were derivatized with phenyl isothiocyanate (PITC). The reaction temperature, reaction time, and concentration of PITC used for derivatization were optimized. Chromatographic separation was on a C₁₈ column with a 65:35 (v/v) mixture of 10 mM phosphate buffer, pH 3.6, and acetonitrile as mobile phase. Heptaminol hydrochloride was used as internal standard.     

Standardization of Crude Extract of Neem Seed Kernels (Azadirachta indica A. Juss) and Commercial Neem Based Formulations Using HPTLC and Extended Length Packed-Columns SFC Method

Two chromatographic techniques are described for the separation and quantitative determination of azadirachtin A and B, salannin, and nimbin present in the crude extract of neem seed kernels and commercial neem based formulations. The high performance thin-layer chromatography (HPTLC) separation of markers was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using ethylacetate-benzene (7.0: 3.0, v/v) as mobile phase. The other technique was based on extended length packed column supercritical fluid chromatographic (PC-SFC) separation of the markers using Cyano column (250 mm × 4.6 mm I.D, S-5.0 μ) and Kromasil 100 NH₂ column (250 mm × 4.6 mm I.D, 5.0 μ) connected in series. The detection was carried out using photodiode array detector at 338 K using methanol-modified carbon dioxide (10%) as the mobile phase at flow rate of 2.0 mL min⁻¹. The current study assesses the effect of extending column length during PC-SFC experiment to obtain maximum resolution between a number of unknown components and known markers present in neem seed extracts. Both the chromatographic methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of quantitative determination of markers by means of HPTLC and PC-SFC method.     

Determination of Tandospirone and its Impurities in Drug Formulations by LC-UV and LC-MS

This work describes the determination of tandospirone in bulk drug substance and formulated products by a reversed-phase liquid chromatographic method with UV detection. Chromatographic separation was performed on a C₁₈ column with a mobile phase of a binary mixture of methanol and water (70:30, v/v) delivered at a flow rate of 0.5 mL min^–1 and detection was performed at 243 nm. The proposed LC method is selective, precise and accurate for the determination of tandospirone in the presence of its manufacturing impurities with a limit of quantitation of 0.54 g mL^–1. A preliminary study for the identification of the major manufacturing impurities was made by liquid chromatography-mass spectrometry with electrospray ionization source operated in a positive ion mode.     

RP-HPLC Determination of Famotidine and its Potential Impurities in Pharmaceuticals

A simple, sensitive, and rapid reversed-phase high-performance liquid chromatographic method has been developed for determination of famotidine (FMT) and its impurities in pharmaceutical formulations. Separations were performed on a Supelcosil LC18 column with an isocratic mobile phase—13:87 (v/v) acetonitrile–0.1 M dihydrogen phosphate buffer containing 0.2% triethylamine (pH 3.0). The mobile phase flow rate was 1 mL min^–1 and the detection wavelength was 265 nm. Response was linearly dependent on concentration between 1 and 80 g mL^–1 (regression coefficient, R², from 0.9981 to 0.9999). RSD from determination of method repeatability (intraday) and reproducibility (interday) were <2% (n=6). Lowest detectable concentrations ranged from 0.08 to 0.14 g mL^–1. The proposed liquid chromatographic method can be satisfactorily used for routine quality control of famotidine in pharmaceutical formulations.     

Analysis of Zoledronic Acid and Its Related Substances by Ion-Pair RP-LC

A simple high-performance liquid chromatographic method – ion-pair reversed- phase high performance liquid chromatography (RPIC) has been developed and employed for the analysis of zoledronic acid and its related substances in bulk material and commercial dosage forms. The mobile phase was a mixture of methanol (20%) and 5 mmol L^–1 phosphate buffer (80%) containing 6 mmol L^–1 tetrabutylammonium bromide, adjusted to pH 7.0 with sodium hydroxide. C₈ column was used as the stationary phase. The chromatographic conditions were optimized. The active ingredient – zoledronic acid was successfully separated from its related substances, including remained imidazol-1-yiacetic acid in the synthesis of zoledronic acid and other possible impurities of oxidation and decomposition. The excipients did not interfere with the determination of zoledronic acid in commercial dosage formulations. The method was rapid, simple, accurate and reproducible. It was not only successfully employed for the assay of zoledronic acid in bulk material and pharmaceutical dosage forms but also for the determination of its related substances.     

Simultaneous HPTLC Determination of Escitalopram Oxalate and Clonazepam in Combined Tablets

A new, simple high-performance thin-layer chromatographic method has been established and validated for simultaneous determination of escitalopram oxalate and clonazepam in a combined tablet dosage form. The drugs were separated on aluminum plates precoated with silica gel 60 F₂₅₄; toluene–ethyl acetate–triethylamine 7:3.5:3 (v/v) was used as mobile phase. Quantitative analysis was performed by densitometric scanning at 258 nm. The method was validated for linearity, accuracy, precision, and robustness. The calibration plot was linear over the ranges 250–2,500 and 50–500 ng band⁻¹ for escitalopram oxalate and clonazepam, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.     

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.     

Stability Indicating HPTLC and LC Determination of Dasatinib in Pharmaceutical Dosage Form

Two sensitive and reproducible methods are described for the quantitative determination of dasatinib in the presence of its degradation products. The first method was based on high performance thin layer chromatography (HPTLC) followed by densitometric measurements of their spots at 280 nm. The separation was on HPTLC aluminium sheets of silica gel 60 F₂₅₄ using toluene:chloroform (7.0:3.0, v/v). This system was found to give compact spots for dasatinib after development (R _F value of 0.23 ± 0.02). The second method was based on high performance liquid chromatography (HPLC) of the drug from its degradation products on reversed phase, PerfectSil column [C₁₈ (5 μm, 25 cm × 4.6 mm, i.d.)] at ambient temperature using mobile phase consisting of methanol:20 mM ammonium acetate with acetic acid (45:55, v/v) pH 3.0 and retention time (t _R = 8.23 ± 0.02 min). Both separation methods were validated as per the ICH guidelines. No chromatographic interference from the tablet excipients was found. Dasatinib was subjected to acid–alkali hydrolysis, oxidation, dry heat, wet heat and photo-degradation. The drug was susceptible to acid–alkali hydrolysis and oxidation. The drug was found to be stable in neutral, wet heat, dry heat and photo-degradation conditions. As the proposed analytical methods could effectively separate the drug from its degradation products, they can be employed as stability indicating.     

Simultaneous determination of azelaic and benzoic acids in topical preparations by liquid chromatography

Summary An isocratic, reversed-phase liquid chromatographic (LC) method has been developed for the simultaneous determination of azelaic and benzoic acids in pharmaceutical creams. The compounds were separated on a C₁₈ column (4 μm particles); the mobile phase was methanolwater, 40∶60, containing 10mm ammonium acetate and with the pH adjusted to 5.0. Detection was performed at 220 nm. The method was validated for accuracy, linearity, precision, and selectivity. Recoveries at levels corresponding to 80% to 120% of the declared content of the creams ranged from 99.5 to 101.8% and from 100.4 to 102.1% for azelaic and benzoic acids, respectively. The calibration graphs were linear in the ranges 20–1400 μg mL⁻¹ for azelaic acid (correlation coefficient,r ₁>0.99999), and 0.1–7.0 μg mL⁻¹ for benzoic acid (r>0.99998).     

Simultaneous Determination of Bifonazole and Benzyl Alcohol in Pharmaceutical Formulations by Reverse-Phase HPLC

A simple, precise and sensitive reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitation of bifonazole, an imidazole antifungal, simultaneously with benzyl alcohol, used as preservative, in pharmaceutical formulations. Method employed Zorbax Eclipse XDB-C18 (250×4.6 mm i.d., 5 m) column, methanol - ammonium acetate (pH 2; 65 mM) (65:35, v/v, pH^* 3.6) as mobile phase with flow rate of 1 mL min^–1 and variable UV detection at 220 and 252 nm. The proposed method was validated by testing its linearity, selectivity, recovery, repeatability, LOD/LOQ values and it was successfully employed for the determination of bifonazole and benzyl alcohol in pharmaceutical cream-based formulations.     

Evaluation of N-Substituted 2,4-Dihydroxyphenylthioamide Fungicide Lipophilicity Using the Chromatographic Techniques HPLC and HPTLC

2,4-Dihydroxyphenylthioamide derivatives modified on the N-aryl ring have substantial fungicidal activity. To determine their quantitative structure–activity relationships their lipophilicity was determined by use of the chromatographic methods column liquid chromatography and thin-layer chromatography. Methanol–water systems were used as mobile phases and the linear dependences of retention (R_M and log k) on volume fraction of organic modifier, φ, were determined. This enabled precise determination of lipophilicity (R_Mw and log k_w) by extrapolation. Correlations were found between quantities characterizing the lipophilicity of the compounds. Deviations enabled discovery of compound structural features which increase or reduce lipophilicity. When these data were correlated with biological activity against the phytopathogenic fungi Alternaria alternata and Botrytis cinerea parabolic dependences were obtained.     

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.     

Stability-Indicating LC Determination of Nitazoxanide in Bulk Drug and in Pharmaceutical Dosage Form

A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for quantitative determination of nitazoxanide in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced decomposition studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5- C18 (250 mm × 4.6 mm, 5 μm) column, and 50 mM ammonium acetate (pH 5.5 by acetic acid) and acetonitrile (55:45 v/v) as a mobile phase. The detection was carried out at a wavelength of 240 nm. The nitazoxanide was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for nitazoxanide in base, acid and in 30% H₂O₂ conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of nitazoxanide was from (100.55 to 101.25%) in the pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, specificity and robustness. The forced degradation studies prove the stability indicating power of the method.     

Application of Multivariate Calibration Techniques to HPLC Data for Quantitative Analysis of a Binary Mixture of Hydrochlorathiazide and Losartan in Tablets

New multivariate approaches have been applied to high-performance liquid chromatography (HPLC) with multiwavelength photodiode-array (PDA) detection. Multivariate calibration techniques such as partial least squares (PLS), principal component regression (PCR), classical least squares (CLS), and inverse least squares (ILS) was subjected to HPLC data for simultaneous quantitative analysis of synthetic binary mixtures and a commercial tablet formulation containing hydrochlorothiazide (HCT) and losartan potassium (LST). The combined use of HPLC and multivariate calibrations has been denoted HPLC–CLS, HPLC–ILS, HPLC–PCR, and HPLC–PLS. Successful chromatographic separation of the two active compounds and enalapril maleate, used as internal standard (IS), was accomplished by means of a 4.6 mm i.d. × 250 mm, 5 m particle, Waters Symmetry C₁₈ reversed-phase column and a mobile phase consisting of 60:40 acetate buffer (0.2 M, pH 4.8)–acetonitrile (v/v, 60:40). HPLC data based on the ratio of analyte peak areas to IS peak area were obtained by PDA detection at five-wavelengths (250, 255, 260, 265, and 270 nm). The HPLC–CLS, HPLC–ILS, HPLC–PCR, and HPLC–PLS calibration plots for hydrochlorothiazide and losartan potassium were constructed separately by using the peak-area ratios corresponding to the concentrations of each active compound. The HPLC multivariate calibrations obtained were tested for different synthetic mixtures containing HCT and LST in the presence of the IS. These multivariate chromatographic methods were also applied to a commercial pharmaceutical dosage form containing HCT and LST. The results obtained from the multivariate calibrations were compared with those obtained by use of another, classical HPLC method using single-wavelength detection.Revised: 29 September 2004 and 4 January 2005