Study of retention parameters obtained in RP-TLC system and their application on QSAR/QSPR of some alpha adrenergic and imidazoline receptor ligands.

The retention constant (R(0)(m)) is determined for 11 selected adrenergic and imidazoline receptor ligands by reverse-phase-thin layer chromatography. It is established that the retention behavior of investigated compounds mostly depends on geometrical, electrostatic, and hydrogen bonding properties. Good correlations among hydrophobic parameters R(0)(m) versus log P for all eleven tested compounds are obtained. The satisfactory correlations are found between R(0)(m) versus apparent partition coefficient octanol-buffer pH 7.4 (log P') or apparent partition coefficient in four liposome systems (log K'(M)) and hypotensive activity (pC(25)) for five imidazolines. The results confirm the suitability of this parameter in quantitative structure-property and structure-activity relationships studies of these drugs.

Use of biopartitioning micellar chromatography and RP-HPLC for the determination of blood-brain barrier penetration of α-adrenergic/imidazoline receptor ligands, and QSPR analysis.

For this study, 31 compounds, including 16 imidazoline/α-adrenergic receptor (IRs/α-ARs) ligands and 15 central nervous system (CNS) drugs, were characterized in terms of the retention factors (k) obtained using biopartitioning micellar and classical reversed phase chromatography (log kBMC and log kwRP, respectively). Based on the retention factor (log kwRP) and slope of the linear curve (S) the isocratic parameter (φ0) was calculated. Obtained retention factors were correlated with experimental log BB values for the group of examined compounds. High correlations were obtained between logarithm of biopartitioning micellar chromatography (BMC) retention factor and effective permeability (r(log kBMC/log BB): 0.77), while for RP-HPLC system the correlations were lower (r(log kwRP/log BB): 0.58; r(S/log BB): -0.50; r(φ0/Pe): 0.61). Based on the log kBMC retention data and calculated molecular parameters of the examined compounds, quantitative structure-permeability relationship (QSPR) models were developed using partial least squares, stepwise multiple linear regression, support vector machine and artificial neural network methodologies. A high degree of structural diversity of the analysed IRs/α-ARs ligands and CNS drugs provides wide applicability domain of the QSPR models for estimation of blood-brain barrier penetration of the related compounds.

Polypharmacology of dopamine receptor ligands.

Most neurological diseases have a multifactorial nature and the number of molecular mechanisms discovered as underpinning these diseases is continuously evolving. The old concept of developing selective agents for a single target does not fit with the medical need of most neurological diseases. The development of designed multiple ligands holds great promises and appears as the next step in drug development for the treatment of these multifactorial diseases. Dopamine and its five receptor subtypes are intimately involved in numerous neurological disorders. Dopamine receptor ligands display a high degree of cross interactions with many other targets including G-protein coupled receptors, transporters, enzymes and ion channels. For brain disorders like Parkinsons disease, schizophrenia and depression the dopaminergic system, being intertwined with many other signaling systems, plays a key role in pathogenesis and therapy. The concept of designed multiple ligands and polypharmacology, which perfectly meets the therapeutic needs for these brain disorders, is herein discussed as a general ligand-based concept while focusing on dopaminergic agents and receptor subtypes in particular.

Determination of some insect repellents in cosmetic products by high-performance thin-layer chromatography.

A simple and reliable thin-layer chromatographic method for the determination of N,N-diethyl-m-toluamide (DEET) and dimethyl phthalate (DMP) in raw material and cosmetic products was developed and validated. A benzene-diethyl ether-cyclohexane (5:3:2, v/v/v) solvent system was used for quantitative evaluation of chromatograms. The chromatographic zones corresponding to the spots of DEET and DMP on the silica gel plates were scanned in the reflectance/absorbance mode at 230 nm. The method was found to be reproducible and convenient for the quantitative analysis of DEET and DMF in raw material and cosmetic products.

High-performance liquid chromatographic assay of 8-hydroxyquinoline sulfate and its stability in immunobiological preparations.

Because of the ability of 8-hydroxyquinoline sulfate (8-HQS) to irreversibly bind metals from rubber stoppers, the stability of 8-HQS in tuberculin solutions was investigated. For the determination of 8-HQS, a simple and sensitive reversed-phase HPLC method with detection at 240 nm was developed and validated. Rapid decreases in concentrations of 8-HQS were found in samples stored in original vials which were exposed to different temperatures and vial positions.

High-performance liquid chromatographic method for the assay of dexamethasone and xylometazoline in nasal drops containing methyl p-hydroxybenzoate.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the determination of dexamethasone sodium phosphate (DSP), xylometazoline hydrochloride (XMC) and methyl p-hydroxybenzoate (MHB). An assay of the compounds has been performed on a HPLC system GBC 1210, at controlled room temperature, on a Nucleosil C8 column (250x3 mm, 5 microm). The mobile phase was acetonitrile-water (35:65, v/v), at a flow-rate of 1 ml min(-1). The parameters for validation such as linearity (r>0.9996), precision (RSD: 0.51-(1.93%), limit of detection and quantification (2.032 x 10(-4) and 4.063 x 10(-4) mg ml(-1) for DSP, 9.7 x 10(-5) and 1.953 x 10(-4) mg ml(-1) for XMC, 1.953 x 10(-4) and 3.096 x 10(-4) mg ml(-1) for MHB) have also been reported. The method was applied to the determination of DSP, XMC and MHB in nasal drops. The statistical parameters were found to be satisfactory, with recovery values ranging from 98.69 to 101.60% (RSD: 0.32-1.03%). The method is simple and accurate and therefore suitable for the simultaneous determination of these compounds in dosage form.

Fluorodensitometric determination of conjugated estrogens in raw material and in pharmaceutical preparations.

A simple, rapid and reproducible fluorodensitometric method for the determination of conjugated estrogens has been developed. The proposed procedure includes the following steps: extraction, hydrolysis of sodium sulphate esters of estrone, equilin, equilenin and their 17-alpha-hydroxy derivatives, separation of the liberated 3-phenolic steroids and in situ measurement of fluorescence. The fluorescence emission was measured after spraying the spots of estrone and estradiol with 2,4-dinitrophenyl-hydrazine in sulphuric acid-ethanol medium and equilin and 17-alpha-dihydroequilin with phosphoric acid and sodium hydroxide solution, respectively. Equilenin and 17-alpha-dihydroequilenin were determined by measuring the native fluorescence. The method applied to the determination of raw material and tablets provided results which agreed well with the stated content and the requirements of USP XXI for conjugated estrogens.

Determination of bifonazole in creams containing methyl- and propyl p-hydroxybenzoate by derivative spectrophotometric method.

A second order derivative spectrophotometric method for the determination of bifonazole in the presence of methyl- and propyl p-hydroxybenzoate as preservatives has been developed. The determination was performed in a 0.1 M HCl solution at 241.5 nm, a wavelength corresponding to the intersection of the second order derivative spectra (2D) of methyl- and propyl p-hydroxybenzoate with the axis (zero-crossing point). On the basis of the knowledge of acidity constants and solubility, as well as of the investigations of zero-order and 2D spectra of bifonazole and preservatives, these conditions were chosen as optimal ones. A calibration curve constructed for bifonazole concentrations ranging from 1.5 to 15 microg/ml had a correlation coefficient of 0.9998. Reliability and reproducibility of the method was checked by analyzing laboratory mixtures of bifonazole and preservatives (recovery 99.97-102.7%; RDS 0.48-1.46%). The proposed method was applied for the determination of bifonazole in a commercial cream formulation. The mean value of bifonazole obtained per 100 g cream was 1.029 g (102.9% of the labeled claim) with a RSD of 0.60%.

Densitometric determination of metoprolol tartrate in pharmaceutical dosage forms.

This paper describes a simple densitometric method for the determination of metroprolol tartrate in tablets and ampoules. After separation on silica gel GF254 plates, using acetone-methanol-triethylamine as the mobile phase for the tablets and acetone-triethylamine for ampoules, the chromatographic zones corresponding to the spots of metoprolol were scanned. Quantitation was performed using a computer-controlled Camag TLC scanner and applying five-point calibration with polynomial regression. The calibration function was established in the ranges 1-28 micrograms for tablets and 1-9 micrograms for ampoules. The results obtained are precise and reproducible, with recovery values of 99.1-99.4%.

Simultaneous HPLC determination of nitrendipine and impurities of the process of synthesis.

A method has been developed for separation of nitrendipine and its impurities of reaction partners and side reaction products by high-performance liquid chromatographic method on a RP-18 column and detection at 238 nm. The mobile phase composition that provided an acceptable nitrendipine resolution, in large excess and possible impurities, in a short elution time, is methanol:water (70:30) and pH 3. Linearity (r> or =0.999), reproducibility (RSD=0.8--1.4%), determination limit (0.5--2%) and recovery (99.8--102.3) were validated and found to be satisfactory. This method enables monitoring of the process of synthesis, as well as the choice of the synthetic design.

Densitometric determination of conjugated oestrogens in the raw material and in pharmaceutical preparations.

A densitometric method for determination of complex mixtures of conjugated oestrogens in raw material and tablets was developed. The proposed procedure comprised hydrolysis of sodium sulphate esters of oestrone, equilin, 17 alpha-oestradiol, equilenin, 17 alpha-dihydroequilin and 17 alpha-dihydroequilenin, the chloroform extraction of free oestrogens and methyltestosterone (internal standard) and separation on TLC plates using chloroform-cyclohexane-dioxane-triethylamine (4.5:4.1:0.6, v/v) as the solvent for development. Quantitative assay was achieved by direct scanning of the oestrogen spots at 280 nm. The proposed method is simple, rapid, reproducible and adequate to control the content of conjugated oestrogens in the raw material and in pharmaceutical preparations.

First-order UV-derivative spectrophotometry in the analysis of omeprazole and pantoprazole sodium salt and corresponding impurities.

The first-order UV-derivative spectrophotometry, applying zero-crossing method was developed for the determination of omeprazole (OM), omeprazole sulphone (OMS), pantoprazole sodium salt (PANa), and N-methylpantoprazole (NPA) in methanol-ammonia 4.0% v/v, where the sufficient spectra resolutions of drug and corresponding impurity were obtained, using the amplitudes 1D(304), 1D(307), 1D(291.5) and 1D(296.5), respectively. Method showed good linearity in the ranges (microg ml(-1)): 1.61-17.2 for OM; 2.15-21.50 for OMS; 2.13-21.30 for PANa and 2.0-20.0 for NPA, accuracy and precision (repeatability and reproducibility). The experimentally determined values of LOD (microg ml(-1)) were 1.126; 0.76; 0.691 and 0.716 for OM, OMS, PANa and NPA, respectively. The obtained values of 2.91% w/w for OMS and 3.58% w/w for NPA in the presence of their parent drug, by applying the method of standard additions, point out the usage of the proposed method in stability studies. Zero-crossing method in the first-order derivative spectrophotometry showed the impurity-drug intermolecular interactions, due to the possible intermolecular hydrogen bonds, confirmed by divergences of experimentally obtained amplitudes for impurities OMS and NPA in comparison to expected values according to regression equations of calibration graphs.

HPTLC determination of ceftriaxone, cefixime and cefotaxime in dosage forms.

The objective of this investigation was to develop a HPTLC method for the determination of ceftriaxone, cefixime and cefotaxime, cephalosporins widely used in clinical practice. High performance TLC of cephalosporins was performed on pre-coated silica gel HPTLC plates with concentrating zone (2.5 x 10 cm) by development in mobile phase ethyl acetate-acetone-methanol-water (5:2.5:2.5:1.5 v/v/v/v). A TLC scanner set at 270 nm was used for direct evaluation of the chromatograms in reflectance/absorbance mode. The calibration curves were established as dependence of peak height (linear and polynomial regression) and peak area (polynomial regression) versus ng level (125-500 ng for all cephalosporins investigated). Relative standard deviations obtained from calibration curves was compared. Precision (RSD: 1.12-2.91% (peak height versus ng) and RSD: 1.05-2.75% (peak area versus ng)), and detection limits (ng level) was validated and found to be satisfactory. The method was found to be reproducible and convenient for quantitative analysis of ceftriaxone, cefixime and cefotaxime in their raw materials and their dosage forms.

Spectrophotometric determination of desoximetasone in ointment using 1,4-dihydrazinophthalazine.

The proposed method is based on coloured hydrazone formation with 1,4-dihydrazinophthalazine as a reagent. Heating at 85 degrees C for 2 h was found necessary to ensure optimal hydrazone formation in the presence of hydrochloric acid. The yellow hydrazone product has an absorption maximum at 380 nm. A linear relationship between absorbance and concentration was established in the concentration range 3.19 x 10(-6) -3.19 x 10(-5) mol l-1 (the regression equation was y = 0.013 167 3 + 0.019 025 9x; correlation coefficient r = 0.9991; n = 6). The detection limit was 1.2 micrograms ml-1 (molar absorptivity found was 1.97 x 10(4) l mol-1 cm-1). The reliability of the proposed method was checked at three different concentrations; the relative standard deviation (RSD) varied from 1.03 to 2.01%. The described method applied to the determination of desoximetasone in ointment gave precise and reproducible results; the recovery was 98.55% with RSD = 2.40% (n = 10).

Quantitative analysis of analgoantipyretics in dosage form using planar chromatography.

In the therapy of pain of weaker genesis, frequently used drugs usually represent a mix of analgoantipyretics of different chemical structures, mostly derivatives of salicylic acid, pyrazolone and p-aminophenol as well as derivatives of propionic and acetylsalicylic acid. For the determination of these drugs, different chromatographic methods have been applied, mostly HPLC, due to the the lower polarity (pyrazolones derivatives) and thermolability, as well as nonvolatility of compounds investigated. TLC method, considering advantages which include simplicity, reasonable sensitivity, rapidity, excellent resolving power and low cost has been successfully explored for the determination of analgoantipyretic compounds. The aim of this work was to develop a simple and rapid HPTLC method for the determination of acetylsalicylic acid, paracetamol, caffeine and phenobarbitone in dosage form. The determination of analgoantipyretics were performed on pre-coated HPTLC silica gel plates (10 x 20 cm(2)) by development in the mobile phase dichlormethane-ethyl acetate-cyclohexane-isopropanol-0.1 M HCL-formic acid (9:8:3:1.5:0.2:0.2 v/v/v/v/v/v). Migration distances (68.6+0.2 mm, 54.1+0.1 mm, 36.4+0.14 mm and 85.9+0.11 mm for acetylsalicylic acid, paracetamol, caffeine and phenobarbitone, respectively) with low RSD values (0.13--0.39%) showed a satisfactory reproductivity of the chromatographic system. TLC scanner was used for direct evaluation of the chromatograms in the reflectance/absorbance mode. Established calibration curves (r>0.999), precision (0.3--1.02%) and detection limits, as well as recovery values (96.51--98.1%) were validated and found to be satisfactory. The method was found to be reproducible and convenient for the quantitative analysis of compounds investigated in their dosage forms.

Gas chromatography-mass spectrometry determination of isosorbide 5-mononitrate and related impurities in raw materials and dosage formulations.

A straightforward quantitative method for gas chromatography-mass spectrometry determination of isosorbide 5-mononitrate (IS5MN) and its related impurities such as isosorbide (IS), isosorbide diacetate (ISDA) and isosorbide 2-acetate-5-nitrate (IS2A5N) in raw materials as well as in dosage formulations is developed. The recovery of these materials was found to be 100.4 +/- 2.4, 99.3 +/- 4.7, 97.8 +/- 5.2 and 100.1 +/- 3.1%, while the detection limits were 27.2, 1.26, 1.02 and 0.78 micrograms in dosage formulations for IS5MN, ISDA, IS2A5N, and IS, respectively. The applicability of the method was tested by analysing three different formulations of IS5MN.

Dependence of the renal excretion of theophylline on its plasma concentrations and urine flow rate in asthmatic children.

The dependence of the renal excretion of theophylline on its plasma concentration and urine flow rate has been investigated in asthmatic children of either sex. One group (age 12.25 +/- 0.80, mean +/- s.d. n = 8) was given aminophylline intravenously (i.v.), while another (age 10.00 +/- 3.64 n = 14) was given a sustained release preparation of theophylline orally (single dose and repeated doses). Unchanged drug (11.6% +/- 1.75) was excreted in the urine corresponding to a renal clearance of 10.6 +/- 1.6 mL h-1kg-1. Time dependence of the renal clearance of theophylline was found only after i.v. administration. Dependence of the renal clearance on urine flow rate was found both after i.v. administration and at steady state, but not after a single oral dose of theophylline. After oral administration, renal clearance of theophylline was higher at steady state than after a single dose (0.58 +/- 0.06 L h-1 kg-1 vs 0.23 +/- 0.03 L h-1 kg-1), while urine flow rate was lower (1.1 +/- 0.5 mL min-1 vs 1.8 +/- 0.9 mL min-1). High correlation of theophylline plasma concentration and theophylline excretion rate was obtained in 10 of 14 patients after administration of a single oral dose of the preparation (r = 0.8567 to 0.9830). There was no dose dependence of the renal clearance of the drug either after a single dose, or at steady state.

Quantitative analysis of sulpiride and impurities of 2-aminomethyl-1-ethylpyrrolidine and methyl-5-sulphamoyl-2-methoxybenzoate in pharmaceuticals by high-performance thin-layer chromatography and scanning densitometry.

A simple and reliable thin-layer chromatographic method for determining sulpiride and impurities of 2-aminomethyl-1-ethylpyrrolidine and methyl-5-sulphamoyl-2-methoxybenzoate was developed and validated. A methylene chloride-methanol-ammonia solution (25%; 18 + 2.8 + 0.4, v/v) solvent system is used for separation and quantitative evaluation of chromatograms. The chromatographic plate is first scanned at 240 nm to locate chromatographic zones corresponding to sulpiride and methyl-5-sulphamoyl-2-methoxybenzoate. Then 2-aminomethyl-1-ethylpyrrolidine is derivatized in situ with ninhydrin, and resulting colored spots are measured at 500 nm. The method is reproducible and convenient for quantitative analysis and purity control of sulpiride in its raw material and in its dosage forms.

Spectrophotometric determination of diltiazem in dosage forms.

A simple and reproducible method for determination of diltiazem in bulk and in dosage forms is presented. The method is based on formation of hydroxamic acid, which reacts with iron (III), forming a complex with a maximum absorption at 525 nm. Assay procedure for diltiazem dosage form requires thin-layer chromatographic separation prior to colorimetric analysis.

UV derivative spectrophotometric study of the photochemical degradation of nisoldipine.

The photodecomposition of nisoldipine ((+/-)3-isobutyl-5-methyl-1,4- dihydro-2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3,5-dicarboxylate), whereby its 4-(2-nitrosophenyl) pyridine analogue is obtained as the photolytic product, was investigated under daylight exposure by means of UV derivative spectrophotometry. The optimal instrumental parameters (120 nm/min scan speed; 2 nm slit width; delta gamma = 10 nm and 5 s response time) for analogue derivative spectra were established for amplitudes 1D285 and 2D291 (measured to the baseline) of the nitroso analogue assay, as well as for 1D386 of the parent compound-nisoldipine assay. Using the first-order derivative spectrum, the minimum detectable amount of nitroso analogue in the presence of nisoldipine was equivalent to an impurity level of 5% and by the second-order derivative spectrum, the determination limit was equivalent to an impurity level of 2%. The degradation of nisoldipine followed within 30 days and the calculated maximal degradation rate was 1.6% per day for nisoldipine raw material, but significantly lower values of 0.19 and 0.15% per day were obtained for Nisoldin tablets (10 and 5 mg, respectively).