Determination of ketoprofen in plasma using high-performance liquid chromatography. Comparison with gas--liquid chromatography (author's transl)

A new method of determination of ketoprofen 2-(3-benzoyl phenyl) propionic acid in plasma using high-performance liquid chromatography (HPLC) is described. After extraction by diethyl either in acidic medium, ketoprofen and the internal standard, 2-(4-benzoyl phenyl) butyric acid, are methylated with gaseous diazomethane and their concentrations measured by HPLC using in LiChrosorb Si 60 (5 micrometer) column and dichloromethane-hexane (60:40) as the mobile phase. The absolute retention times of the internal standard and ketoprofen are 11.6 and 12.8 min, respectively. The precision of the methods is +/- 4% and the lower detection limit ranges from 0.06 to 0.10 microgram/ml. The results obtained by HPLC show a very good correlation with those obtained by gas--liquid chromatography. The proposed method is sensitive, reproducible and rapid and very suitable for ketoprofen determination in pharmacokinetic studies.     

High-performance liquid chromatography with fluorescence detection for the simultaneous determination of 3,4-methylenedioxymethamphetamine, methamphetamine and their metabolites in human hair using DIB-Cl as a label

This paper describes a highly sensitive HPLC method for the simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in human hair samples. The amphetamines investigated were derivatized with the fluorescent reagent, DIB-Cl to yield highly fluorescent DIB-derivatives, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 nm and 430 nm, respectively. The separation was achieved on an ODS column with an isocratic mobile phase composed of acetonitrile-methanol-water (30:40:30, v/v/v). The limits of detection for the four compounds obtained by the proposed method ranged from 11 to 200 pg/mg. The method was successfully applied to the determination of MDMA and MDA in hair samples obtained from MDMA abuser.     

Simultaneous solvent extraction and high-performance liquid chromatographic determination of uranium, iron, nickel and copper in mineral ore samples and phosphate rock residues using N,N′-ethylenebis(salicylaldimine) as complexing reagent

The reagent N,N-ethylenebis(salicylaldimine) (H₂SA₂en) has been examined for precolumn derivatization, followed by solvent extraction in chloroform and HPLC determination of uranium, iron, nickel and copper on a Hypersil ODS 3 μm column. Complexes were eluted isocratically using a ternary mixture of methanol-acetonitrile-water and UV detection was at 260 nm. The detection limit was 12 ng/injection for each of the elements. The method has been applied for the determination of metals in mineral ore samples and phosphate rock residues. The results obtained are compared using atomic absorption.     

Validation and evaluation of a high performance liquid chromatographic method for the determination of aluminium in wine

A new method is reported for the determination of aluminium in wine by HPLC, involving derivatisation with 8-hydroxyquinoline (oxine) in the presence of micelles resulting in the formation of a fluorescent derivative. The complex is separated on a C18 column using a mobile phase of oxine - SDS - 35% acetonitrile, in a pH 7 buffer. The method was validated in the range 0.125-2 mg/l in a synthetic wine. The method was applied to the determination of aluminium in white, rosé and red wines and results compared with those obtained by atomic absorption (GFAA). Aluminium concentrations found by HPLC in white wines were greater than those found in red wines. Further investigation using a polyphenol-enriched white wine revealed a statistically significant inverse relationship between wine polyphenol content and the aluminium concentration determined by HPLC. The method may therefore be envisaged for the determination of unbound aluminium in wine.     

Kinetic-spectrophotometric determination of theophylline,dyphylline, and proxyphylline by use of partial least-squares regression

A kinetic-spectrophotometric method for the determination of theophylline, dyphylline and proxyphylline, based on their azo coupling reaction with the diazonium ion of sulfanilic acid after a treatment with alkali, is proposed. The absorbance is recorded from 340 to 600 nm every second during reaction for 90 s, and calibration is performed by partial least-squares regression, using first derivative spectra values. Mixtures containing 2.5-13 micro g mL(-1) dyphylline and proxyphylline, and 2-9 micro g mL(-1) theophylline were successfully resolved with root mean squared errors of prediction (RMSEP) of 0.4, 0.3, and 0.2 for dyphylline, proxyphylline, and theophylline, respectively. The proposed method was satisfactorily applied to the determination of the three compounds in a commercially available pharmaceutical preparation and provided results similar to those obtained by HPLC.     

High-performance liquid chromatographic determination of loracarbef, a potential metabolite, cefaclor and cephalexin in human plasma, serum and urine

A high-performance liquid chromatographic (HPLC) method is reported for the determination of a new carbacephem antibiotic, loracarbef, a hydroxylated analogue, and two cephalosporins, cefaclor and cephalexin, in plasma, serum, and urine. The antibiotics are extracted from plasma by means of C18 solid-phase cartridges. Urine samples are diluted with water and directly injected on the HPLC system. The HPLC system utilizes a Supelcosil LC-18-DB (250 mm x 4.6 mm I.D.) reversed-phase column and ultraviolet detection at 265 nm. The limit of quantitation is 0.5 micrograms/ml for each compound. Excellent correlation of plasma concentrations is shown between results determined by HPLC and those obtained by microbiological agar-well diffusion assays. Stability studies of loracarbef in human plasma show the antibiotic to be stable for at least 24 h at room temperature and for at least twelve months at -20 degrees C.     

Determination of acetaldehyde in human blood by high-performance liquid chromatography using fluorometry

A method was developed for the measurement of acetaldehyde in human blood by high-performance liquid chromatography (HPLC). The method was based on a pre-column reaction; a fluorescent substance was formed by a coupling reaction between 2 mol of cyclohexan-1,3-dione and 1 mol of acetaldehyde with ammonium acetate. The coupling compound was analyzed by HPLC and the concentration of acetaldehyde was obtained from the calibration curve drawn from the results using standard solutions. The determination was sensitive and reproducible with a range of 0.2-10 microM and a precision (coefficient of variation) of 2.43%.     

Determination of alkyl sulfates in the influent and effluent of a laboratory activated sludge plant by HPLC

Summary A method for the determination of alkyl sulfates by HPLC is described. The method is based on the decomposition of the alkyl sulfates by sulfuric acid, extraction of the resulting alkyl alcohols, derivatization of the alcohols by phenyl isocyanate, and reversed-phase HPLC with UV-detection. The described method is well suited to investigate the biodegradability of alkyl sulfates in laboratory activated sludge plants. An advantage of the HPLC procedure in comparison to the determination of methylene-blue-active substances (MBAS) is that informations about the kinetics of the biodegradation, the structure of substances and possible metabolites can be obtained.     

Simultaneous spectrophotometric determination of carbamazepine and phenytoin in serum by PLS regression and comparison with HPLC

Carbamazepine (CBZ) and phenytoin (PHT) are two antiepileptic drugs which are used simultaneously. In this paper a partial least-squares (PLS) calibration method is described for the simultaneous spectrophotometric determination of CBZ and PHT in plasma. Standard binary mixtures of CBZ and PHT have been resolved by application of PLS-1 to their UV spectra. Then, the binary standard solutions, spiked to plasma, were prepared and after the extraction of the drugs, their corresponding UV spectrum were analyzed by PLS regression to calculate the concentration of drugs in unknown plasma. A leave one out cross-validation procedure was employed to find the optimum numbers of latent variables using PRESS. A HPLC method was also applied for simultaneous determination of two drugs in the plasma and in methanol. The mean recoveries obtained by PLS were 98.4 and 98.2 for CBZ and PHT and those obtained by HPLC were 100.1 and 101.7, respectively. Although, the HPLC method showed better performance than PLS, it was found that the results obtained by PLS were comparable with those obtained by HPLC method.     

Determination and validation of duloxetine hydrochloride in capsules by HPLC with pre-column derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of duloxetine hydrochloride in capsules. The method was based on pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole using the fluorimetric detection technique. Duloxetine hydrochloride was analyzed by HPLC using an Inertsil C18 column (5 μm, 150 × 4.6 mm) and mobile phase consisted of methanol and water (65:35, v/v). The fluorescence detector was adjusted at excitation and emission wavelengths of 461 and 521 nm, respectively. The linearity of the method was in the range of 10-600 ng/mL. Limits of detection and quantification were 0.51 and 1.53 ng/mL, respectively. The proposed method was successfully applied for determination of duloxetine hydrochloride in its pharmaceutical preparation. The results were in good agreement with those obtained using a reference method.     

Quantitative analysis of the cholesterol-lowering drugs ezetimibe and simvastatin in pure powder, binary mixtures, and a combined dosage form by spectrophotometry, chemometry, and high-performance column liquid chromatography

Simple, accurate, sensitive, and precise UV spectrophotometric, chemometric, and HPLC methods were developed for simultaneous determination of a two-component drug mixture of ezetimibe (EZ) and simvastatin (SM) in laboratory-prepared mixtures and a combined tablet dosage form. Four spectrophotometric methods were developed, namely, ratio spectra derivative, ratio subtraction, isosbestic point, and mean centering of ratio spectra. The developed chemometric-assisted spectrophotometric method was the concentration residual augmented classical least-squares method; its prediction ability was assessed and compared to the conventional partial least-squares method. The developed HPLC method used an RP ZORBAX C18 column (5 microm particle size, 250 x 4.6 mm id) with isocratic elution. The mobile phase was acetonitrile-pH 3.5 phosphate buffer (40 + 60, v/v) at a flow rate of 1.0 mL/min, with UV detection at 230 nm. The accuracy, precision, and linearity ranges of the developed methods were determined. The developed methods were successfully applied for determination of EZ and SM in bulk powder, laboratory-prepared mixtures, and a combined dosage form. The results obtained were compared statistically with each other and to those of a reported HPLC method; there was no significant difference between the proposed methods and the reported method regarding both accuracy and precision.     

HPLC determination of ibuprofen, diclofenac and salicylic acid using hollow fiber-based liquid phase microextraction (HF-LPME)

This paper describes an extraction method using a polypropylene membrane supporting dihexyl ether (three-phase hollow fiber-based liquid phase microextraction (HF-LPME)) for the analysis of several pharmaceuticals (salicylic acid (SAC), ibuprofen (IBU) and diclofenac (DIC)) followed by a HPLC determination using a monolithic silica type HPLC column, that allows lower retention times than the usual packed columns with adequate resolution. Detection was realized by means of a coupled in series diode array (DAD) and fluorescence (FLD) detectors. HF-LPME is a relatively new technique employed in analytical chemistry for sample pretreatment which offers more selectivity and sensitivity than any traditional extraction technique. Detection limits by DAD are 12, 53 and 40 ng mL⁻¹ for salicylic acid, diclofenac and ibuprofen, respectively and by FLD 7 and 2 ng mL⁻¹ for salicylic acid, and ibuprofen. The method has been successfully applied to their direct determination in human urine and the results obtained demonstrated that could be also applied to the determination of the corresponding metabolites.     

Determination of phenols by liquid chromatography after online adsorption preconcentration on the Strata-X adsorbent

An online adsorption-high-performance liquid chromatography (HPLC) method was developed for the determination of phenols (11 priority water pollutants) in waters, using polystyrene chemically modified with N-vinyl-2-pyrrolidone (Strata-X). The detection limits for phenols obtained using a preconcentration from 10 mL of water and calculated by different means vary from 0.3 to 2 μg/L. The accuracy of the determination of phenols in tap and river water samples was verified by the standard addition method.     

Development of a new high-performance liquid chromatographic method for the determination of ceftazidime

A rapid, accurate, and sensitive high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of ceftazidime in pharmaceuticals. The method validation parameters yielded good results and included range, linearity, precision, accuracy, specificity, and recovery. The excipients in the commercial powder for injection did not interfere with the assay. Reversed-phase chromatography was used for the HPLC separation on a Waters C18 (WAT 054275; Milford, MA) column with methanol-water (70 + 30, v/v) as the mobile phase pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 245 nm. The calibration graph for ceftazidime was linear from 50.0 to 300.0 microg/mL. The values for interday and intraday precision (relative standard deviation) were <1%. The results obtained by the HPLC method were calculated statistically by analysis of variance. We concluded that the HPLC method is satisfactory for the determination of ceftazidime in the raw material and pharmaceuticals.     

Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.     

High-performance liquid chromatographic determination of tocopherols in infant formulas

A method for the simultaneous determination of alpha-tocopherol acetate and alpha-, delta-, and gamma-tocopherols by normal-phase high-performance liquid chromatography (HPLC) with a fluorescent detector in infant formula is proposed. The values obtained in the determination of the analytical parameters: linearity, precision, limit of detection and accuracy (analysis of a standard reference material, SRM 1846), confirm the quality of the method. The proposed method is useful for the determination of alpha-, delta-, and gamma-tocopherols and alpha-tocopherol acetate in infant formulas at a low cost and in a total time of 2 h.     

Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.     

Comparison of second derivative-spectrophotometric and reversed-phase HPLC methods for the determination of prednisolone in pharmaceutical formulations.

Second derivative-spectrophotometric and high-performance liquid chromatographic methods for the determination of prednisolone in pharmaceutical formulations have been developed. Determination of prednisolone in tablets was conducted by using a second-order derivative UV spectrophotometric method at 250 nm (n = 5). Standards for the calibration graph ranging from 5.0 to 35.0 microg/ml were prepared from stock solution. The proposed method was accurate, with 98% recovery value, and precise, with a coefficient of variation (CV) of 1.38. These results were compared with those obtained by an exclusively developed isocratic reversed-phase high-performance liquid chromatography (HPLC) method. An isocratic reversed-phase Bondapak C(18) column with acetonitrile-citrophosphate buffer (pH 5; 45:55 v/v) mobile phase was used and UV detector was set to 241 nm using 11 alpha-hydroxyprogesterone as an internal standard. Calibration solutions used in HPLC were in the range from 2 to 300 microg/ml. Results obtained by derivative UV spectrophotometric method were comparable to those obtained by HPLC method, as far as analysis of variance (ANOVA) test, F(calculated), 0.762 and F(theoretical), 3.89, results were concerned.     

Chemometric determination of naproxen sodium and pseudoephedrine hydrochloride in tablets by HPLC

A new chemometric determination by high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection was implemented for the simultaneous determination of naproxen sodium and pseudoephedrine hydrochloride in tablets. Three chemometric calibration techniques, classical least squares (CLS), principle component regression (PCR) and partial least squares (PLS) were applied to the peak area at multiwavelength PDA detector responses. The combinations of HPLC with chemometric calibration techniques were called HPLC-CLS, HPLC-PCR and HPLC-PLS. For comparison purposes the HPLC method called the classic HPLC method was used to confirm the results obtained from combined HPLC-chemometric calibration techniques. A good chromatographic separation between two drugs with losartan potassium as an internal standard was achieved using a Waters Symmetry C18 Column 5 microm 4.6+/-250 mm and a mobile phase containing 0.2 M acetate buffer and acetonitrile (v/v, 40:60). The multiwavelength PDA detection was measured at five different wavelengths. The chromatograms were recorded as a training set in the mobile phase. Three HPLC-chemometric calibrations and the classic-HPLC method were used to test the synthetic mixtures of naproxen sodium and pseudoephedrine hydrochloride in the presence of the internal standard. The HPLC-chemometric approaches were applied to real samples containing drugs of interest. The experimental results obtained from HPLC-chemometric calibrations were compared with those obtained by a classic HPLC method.     

Simultaneous determination of potassium guaiacolsulfonate, guaifenesin, diphenhydramine HCl and carbetapentane citrate in syrups by using HPLC-DAD coupled with partial least squares multivariate calibration

A simple and rapid analytical procedure was proposed for the determination of chromatographic peaks by means of partial least squares multivariate calibration (PLS) of high-performance liquid chromatography with diode array detection (HPLC-DAD). The method is exemplified with analysis of quaternary mixtures of potassium guaiacolsulfonate (PG), guaifenesin (GU), diphenhydramine HCI (DP) and carbetapentane citrate (CP) in syrup preparations. In this method, the area does not need to be directly measured and predictions are more accurate. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of analytes could be obtained with HPLC-DAD coupled with PLS calibration. This method was tested by analyzing the synthetic mixture of PG, GU, DP and CP. As a comparison method, a classsical HPLC method was used. The proposed methods were applied to syrups samples containing four drugs and the obtained results were statistically compared with each other. Finally, the main advantage of HPLC-PLS method over the classical HPLC method tried to emphasized as the using of simple mobile phase, shorter analysis time and no use of internal standard and gradient elution.