Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics.

HPLC analyses of pharmaceutical dosage forms containing analgesics and related compounds (acetylsalicyclic acid, paracetamol, propyphenazone, caffeine and chlorpheniramine) were performed on C18 and cyano columns under reversed-phase conditions. The performance of the methods was enhanced by introducing postcolumn on-line photochemical derivatization in combination with a diode-array detection. The column effluents were subjected on-line to UV irradiation (254 nm) and the characteristic photo-induced spectral modifications were useful for the unambiguous identification of the various analgesic compounds. The proposed HPLC methods were successfully applied to the analysis of commercially available analgesic dosage forms.

Determination of retinoids in galenicals by column liquid chromatography with fluorescence and diode-array detection.

Simple and rapid reversed-phase gradient column liquid chromatography (LC) with fluorescence detection at different wavelengths was developed for the simultaneous analysis of all-trans, 13-cis, 9-cis retinoic acids, vitamin A palmitate and beta-carotene in galenicals. The assay results agreed with those obtained by an LC method with diode-array UV detection. A post-column on-line photochemical reactor (irradiation at 254 and 366 nm) was inserted between the LC column and the fluorescence detector to enhance the performance of the method. Two fluorescence spectra (photoreactor on and off) were obtained for each analyte which proved useful for the unambiguous identification of the various analytes.

Colorimetric determination of acetylcysteine, penicillamine, and mercaptopropionylglycine in pharmaceutical dosage forms.

A colorimetric determination of acetylcysteine, penicillamine, and mercaptopropionylglycine is described. The method is based on the oxidation of mercapto-compounds with iron(III) in the presence of 1,10-phenanthroline. The iron(II) formed was quantitatively and rapidly converted to the stable tris(1,10-phenanthroline)iron(II) complex measured spectrophotometrically at 515 nm. The results obtained from various commercial formulations indicate that the method proposed allows a simple, sensitive determination of these mercapto-drugs with good accuracy (99.5-101% recovery) and remarkable precision (RSD +/- 0.6-1.6%).

Analysis of miconazole and econazole in pharmaceutical formulations by derivative UV spectroscopy and liquid chromatography (HPLC).

Methods based on derivative UV spectrophotometry and high-performance liquid chromatography (HPLC) have been developed for the selective determination of miconazole and econazole in pharmaceutical dosage forms. A solid-phase extraction (SPE) procedure using a diol column gave quantitative drug extraction from formulated creams and provided purified sample solutions suitable for assay by the derivative UV spectrophotometric and HPLC methods. The proposed methods gave comparable accurate results, whereas a conventional UV spectrophotometric method was found to be seriously affected by excipients.

Analysis of flucytosine dosage forms by derivative UV spectroscopy and liquid chromatography.

A simple second-order derivative spectrophotometric method was developed for the selective determination of flucytosine (an antimycotic drug) in the presence of 5-fluorouracil (a cytotoxic agent), its synthetic precursor and degradation product. Traces of 5-fluorouracil in flucytosine were also determined by derivative UV spectroscopy; flucytosine was removed by a selective solid-phase extraction (SPE) procedure using a strong cation-exchange sorbent. The spectrophotometric methods were applied successfully to the quality control of commercial dosage forms of flucytosine and the results were compared with those obtained by a HPLC procedure (cyano column) developed as a reference method.

Analysis of nandrolone esters in oily injections by reversed-phase high-performance liquid chromatography.

A specific liquid chromatographic (HPLC) procedure is presented for the analysis of nandrolone esters (phenylpropionate, decanoate and undecanoate) in commercial oily injections. The analysis was carried out under isocratic, reversed-phase (RP8 column) conditions using a UV detector (240 nm). The system discriminates between nandrolone alcohol, a potential impurity, and its esters and permits the quantitation of trace benzaldehyde derived from the oxidative degradation of benzyl alcohol. The proposed HPLC method was found to be more specific and accurate than the pharmacopoeial spectrophotometric assay procedure (isoniazid reagent). The interference of benzaldehyde with the isoniazid method was also investigated.

Validation of a spectrophotometric method for the determination of iron (III) impurities in dosage forms.

A spectrophotometric method (lambda=535 nm) for the iron (III) impurities determination in iron protein-succinylate complex syrup using thioglycolic acid in basic ambient was proposed and validated. Assay samples were treated with 0.1 N hydrochloric acid and centrifuged to remove the interfering active drug. Linear response (r=0.9999) was observed over the range of 0.005-0.2% of the iron (III) with respect to the complex nominal concentration. The accuracy could be considered very satisfactory (recovery=97-99%). The intra-day precision (RSD) of impurity amongst six independent sample preparations, was 1.4%, and there was no significant difference between intra- and inter-day studies. Intermediate precision indicated that the assay possessed high degrees of ruggedness. The limit of quantitation was 0.005% of impurity with respect to the active drug. The results obtained for iron (III) were compared statistically with those obtained with the standard addition method by means of the Student's t-test and the variance ratio F-test; no significative difference was found.

GC-MS analysis of incenses for possible presence of allergenic nitromusks.

A Gas chromatographic method with mass detector was developed to identify and determine nitromusks in incense sticks of different origin (India, China, Tibet). The proposed method was found useful to correlate dermatological allergic reactions with the use and composition of commercial incense sticks. The incense sticks were powdered, extracted with methanol and after the addition of 1-eicosanol as internal standard, injected into the GC-MS, using 25 m bonded phase fused capillary column methyl, 5% phenyl silicone (0.32 mm I.D., 0.25 microns film thickness). Musk ambrette was identified and determined in one kind of chinese incense together with musk ketone and musk xylene. The latter compound was also found alone in another kind of chinese incense.

HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations.

Reversed-phase HPLC on different column packing materials (Hypersil C-18, Spherisorb-CN, Chromspher-B) is used to obtain selective separations of imidazole antimycotic drugs, such as ketoconazole, clotrimazole, tioconazole, bifonazole, isoconazole, econazole, miconazole and fenticonazole. The use of a post-column on-line photochemical reactor is shown to be useful for the enhancement of the sensitivity of the HPLC analysis with UV detection. The proposed HPLC methods are applied to the analysis of commercial dosage forms (creams) with solid-phase extraction (SPE) procedure, using a diol sorbent, being found to be a convenient technique for the sample preparation giving quantitative drug recovery.

HPLC-fluorescence determination of unconjugated estrogens in pharmaceuticals.

A fluorimetric liquid chromatographic method (lambda(ex) = 280 nm; lambda(em) = 312 nm) was developed for measurements of unconjugated estrogens (estradiol and estriol) in pharmaceutical dosage forms using a reversed-phase column with water acetonitrile at different composition as mobile phase. The in vitro release profiles of three different estradiol transdermal therapeutic systems were determined through a medical-grade silicone rubber subdermal implant material membrane, using a modified Franz diffusion apparatus at 37 degrees C in presence of PEG 400. The HPLC method possesses advantages of rapidity, simplicity and accuracy.

On-line post-column photochemical derivatization in liquid chromatographic--diode-array detection analysis of binary drug mixtures.

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.

Analysis of aliphatic dicarboxylic acids in pharmaceuticals and cosmetics by liquid chromatography (HPLC) with fluorescence detection.

2-Bromoacetyl-6-methoxynaphthalene has been found to be a useful prechromatographic fluorescent labelling reagent for the analysis of dicarboxylic acids. The derivatization reaction of azelaic acid and meglutol with this reagent yieded stable and highly fluorescent diesters which could be analysed by reversed-phase HPLC with fluorescence detection. According to the nature of the sample, the derivatization reaction could be carried out in acetonitrile or in an aqueous micellar system. The proposed methods proved to be suitable for the quality control of various complex pharmaceutical and cosmetic formulations of the azelaic acid and meglutol.

HPLC determination of glutathione and L-cysteine in pharmaceuticals after derivatization with ethacrynic acid.

Ethacrynic acid and its methyl ester are proposed as useful pre-chromatographic derivatization reagents for the HPLC analysis (UV detection) of reduced glutathione (GSH) and L-cysteine. The optimum experimental conditions for the thiol derivatization, the removal of the excess reagent by liquid-liquid or solid-phase extraction and the reversed-phase chromatographic separations of the thiol adducts were investigated. The method was applied to the HPLC determination of GSH and L-cysteine in commercial formulations and proved to be suitable for the HPLC determination of oxidized glutathione (GSSG) after reduction to GSH using dithiothreitol (DTT).

Analysis of amphetamine and congeners in illicit samples by liquid chromatography and capillary electrophoresis.

Screening methods based on liquid chromatography (HPLC) and capillary electrophoresis (CE) have been developed for the identification and determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine in illicit tablets. Diethylpropione, (-)-ephedrine, and 3-amino-1-phenylbutane were also included in the study as amphetamino-related compounds. The HPLC-diode-array detection method involved on-line photochemical derivatization to enhance the selectivity of detection allowing amphetamines to be distinguished from related compounds such as diethylpropione (amfepramone). When the CE approach was adopted, two identification parameters (UV spectra and migration index) were used and the enantioresolution of the racemic amphetamines was achieved using hydroxypropyl-beta-cyclodextrin as chiral selector.

Crosslinked poly(methyl vinyl ether-co-maleic anhydride) as topical vehicles for hydrophilic and lipophilic drugs.

Poly(methyl vinyl ether-co-maleic anhydride) crosslinked with ethylene glycol (GZ-ET), 1,4-butanediol (GZ-BUT), 1,6-exandiol (GZ-EX), 1,8-octanediol (GZ-OCT), 1,10-decanediol (GZ-DEC) or 1,12-dodecanediol (GZ-DOD) was prepared and employed as a supporting material for aqueous topical gels containing pyridoxine hydrochloride (PYCL) chosen as a hydrophilic model molecule or for O/A emulsion containing beta-carotene chosen as a hydrophobic model molecule. We analyzed the effect of the nature of the crosslinker on the permeation of hydrophilic and lipophilic vitamins through porcine skin by in vitro permeation studies. The vehicles formed by crosslinked poly(methyl vinyl ether-co-maleic anhydride) showed enhanced vitamins permeation with respect to the same vehicles formed by noncrosslinked poly(methyl vinyl ether-co-maleic anhydride) (GZ). The decrease in the crosslinker acyl chain length provides vehicles accelerating the drug permeability through the skin.

Determination of salicilic acid in saliva by high-performance liquid chromatography.

A sensitive reversed-phase HPLC method with fluorimetric detection was developed for the determination of salicilic acid in saliva samples. The method, involving a preliminary solid-phase extraction procedure, was found to be suitable for the determination of salivary salicilic acid concentrations following the administration of a single oral dose of some commercial acetylsalicilic acid formulations.

Determination of minoxidil in pharmaceutical formulations by difference spectrophotometry and liquid chromatography (HPLC).

A simple difference spectrophotometric method has been developed for the selective and rapid determination of minoxidil in pharmaceutical formulations. The method is based on the characteristic modifications of the minoxidil UV spectrum induced by pH changes; a difference spectrum can be obtained and the absorbance A282.8 or the absorbance difference delta A = (A282.8-A259.2) can be used for the selective drug quantitation. As an alternative, a derivative second-order spectrophotometric method was also developed. The spectrophotometric methods were applied to the quality control of commercial minoxidil dosage forms and the results were comparable with those obtained by a reference HPLC procedure.

Analysis of Trimethoprim--sulfonamide drug combinations in dosage forms by UV spectroscopy and liquid chromatography (HPLC).

Derivative and difference spectrophotometric methods are described for the direct simultaneous analysis of combinations of Trimethoprim with sulfonamide drugs (sulfadiazine, sulfamethoxazole, sulfamethoxypyridazine) in commercial dosage forms. The more advantageous approach (derivative and difference mode) is suggested for each binary mixture. The assay results are compared with those obtained by a new chromatographic (HPLC) method, involving a C-18 column and a mobile phase (pH 6.5) containing 1.8 diaminooctane as amine modifier and sodium heptansulfonate as ion pairing agent.