Rita Porrà

Development of a tandem thin-layer chromatography–high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products

Abstract A solid-phase extraction clean-up and and a liquid chromatographic method with ultraviolet detection were developed for the analysis of 51 corticosteroids in cosmetic samples in order to screen commercial samples for the presence of undeclared synthetic corticosteroids. A thin-layer chromatographic analysis was carried out on silica gel plates, using different eluants and detection reagents. When such a preliminary chromatographic separation gave some indications about the presence of steroid compounds, the methanol extracts from real samples were applied to a solid-phase extraction C 18 cartridge, and the analytes eluted with ethyl ether. The high-performance liquid chromatographic separation was then carried out for the identification and determination of the analytes using a Purospher RP-18 column, an isocratic or a gradient elution with a mixture acetonitrile–water and a photodiode-array detector. The accuracy of the method was determined by spiking experiments on home-made cosmetic samples. The analytical recoveries were satisfactory.

Carotenoids, retinoids and alpha-tocopherol in human serum: Identification and determination by reversed-phase HPLC

A rapid, simple and specific high performance liquid chromatographic procedure for assaying alpha- and beta-carotene is described. The method also enables the simultaneous determination of retinol and dl-alpha-tocopherol in human serum. The same chromatographic procedure can be used to assay the major carotenoids in human serum, provided analyses are replicated and the effluent is monitored at 450 nm. The conditions described also enable determination of licopene, cryptoxanthine and lutein with zeaxanthine. An aliquot of 0.5 ml serum is deproteinized with ethanol (0.5 ml) and extracted with petroleum ether (0.75 ml). The petroleum ether extract is evaporated until dry and then redissolved immediately with 0.5 ml of an eluent mixture consisting of methanol-hexane (85:15, v/v). Aliquots of 50 microl are then injected onto a 250 x 4.6 mm column packed with Spherisorb ODS-2. Owing to its good reproducibility, the procedure can be used for assays with external standards. Clinical applications are described for cases of hypercarotinemia associated with endocrine dysfunctions such as hypothyroidism and diabetes.

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

SummaryCapillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8 % and <1.2 %, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.

Stability of reconstituted solutions of ceftazidime for injections: an HPLC and CE approach

The stability of aqueous reconstituted ceftazidime injection vials containing ceftazidime pentahydrate blended with anhydrous sodium carbonate was investigated in different storage conditions (4 degrees C and 10 degrees C for 7 days in a refrigerator, 20 and 30 degrees C for 24 h) with validated HPLC and (micellar) CE methods. Stability indicating data were obtained for ceftazidime and two degradation products: pyridine and the delta2-ceftazidime isomer. Other degradation products were also identified (the complementarity of the two used experimental procedures was useful in such exercise) and characterized by their UV spectra and retention times. Stability data (7 days at 4 degrees C in a refrigerator and 18 h at room temperature) resulted in agreements with the manufacturers prescription and point out the need of a strict temperature control of the refrigerators compartment used to store the reconstituted solution.

LTB4 as marker of 5-LO inhibitory activity of two new N-ω-ethoxycarbonyl-4-quinolones

The supposed 5-LO inhibitory activity of two N-ω-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from celle cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Analysis of ceftazidime and related compounds by micellar electrokinetic chromatography

A micellar electrokinetic chromatographic method for the separation and quantification of ceftazidime, its delta2-isomer and pyridine (two ceftazidime related impurities) was developed and validated. Optimised conditions were obtained using an electrolyte system consisting of 25 mM sodium tetraborate, at pH 9.2, and 75 mM sodium dodecylsulphate. A limit of detection of 0.2 microg ml(-1) and a limit of quantitation of 0.6 microg ml(-1) were estimated for pyridine and delta2-isomer: this means that levels of < 0.1% of pyridine and delta2-isomer in ceftazidime can be determined. Calibration curves for all analytes were linear over the studied ranges with correlation coefficients >0.999. Good reproducibility for migration times and corrected peak areas were achieved (RSD % 0.3 and 1.0, respectively). The results demonstrate that the method is reproducible, accurate and appropriate for ceftazidime assay in pharmaceutical samples.

The identification of related substances in 9α-fluoroprednisolone-21 acetate by means of high-performance liquid chromatography with diode array detector and mass spectrometry

A method for the analysis and identification of the principal related substances in 9 alpha-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance. Thus, after reversed-phase chromatographic separation and observation of the UV spectra and their respective second derivatives, it was possible to characterize some of the principal effective and potential related substances such as 9 alpha-fluorohydrocortisone acetate, 9 alpha-bromoprednisolone acetate, 9 beta, 11 beta-epoxyprednisolone acetate and 9(11)-dehydroprednisolone acetate, emerging as chromatographic peaks. Identification of 9 alpha-bromoprednisolone acetate and of 9 alpha-fluorohydrocortisone acetate which proved to be the most significant impurities, was confirmed by means of an exhaustive study of the mass spectra of these substances conveniently isolated by normal-phase HPLC. The chromatographic, spectrophotometric and mass-spectrometric characteristics of the studied compounds are reported.

Screening of preservatives by HPLC-PDA-ESI/MS: A focus on both allowed and recently forbidden compounds in the new EU cosmetics regulation

Commission regulation (EU) No 358/2014 amending the new regulation (EC) No 1223/2009 on cosmetics has prohibited the use of isopropyl-, isobutyl-, phenyl-, benzyl- and pentylparaben. Furthermore, Commission regulation (EU) No 1004/2014 has lowered the maximum permitted concentration of butyl- and propylparaben in cosmetics and it has also banned them in leave-on products designed for application on the nappy area of children under three years of age. A HPLC-PDA-ESI/MS method has been developed herein for the detection of seventeen preservatives, both the most utilised and the recently forbidden by the new EU regulations. The separation of these compounds, including benzoic acid and its derivatives in a 1.10 - 3.04 log Pow range, has been performed with a gradient elution on a Symmetry(®) C18 column (250×4.6mm i.d., particle size 5μm) with water and acetonitrile (0.1% formic acid) as mobile phase. Quantification has been carried out by HPLC-PDA. The method has been validated and successfully applied to the analysis of a large number of cosmetics with different functions like rinse-off and leave-on, or composition like skin, hair, face and oral products.

Identification and determination of corticosteroids in adrenal gland extracts for pharmaceutical use by HPLC

SummarySome HPLC procedures with isocratic or gradient elution are reported for the identification and determination of most of the characteristic components of cortical extracts.The proposed solvent systems were: A) for normal phase chromatography, mixtures of chloroform-methanol-water on silica columns. B) For reversed phase chromatography, mixtures of methanol-water or acetonitrile-water or tetrahydrofuran-water on octadecyl silica columns of different brands. With these systems it was possible to identify and determine, in addition to the principal corticosteroids, some minor components of the cortical extracts as the 20β-dihydroderivatives of compounds F, E, A, B, the 17-ketosteroids adrenosterone, 11β-hydroxyandrostendione and androstendione and finally, progesterone and 17-OH progesterone. In reversed phase chromatography it was also possible, by monitoring the effluent at 205 nm, to reveal the 5α- and 5β-tetrahydroderivatives of the main corticosteroids and to separate them from most of the steroidal components of the adrenal extracts; in these conditions it was also possible to reveal some characteristic, unknown components of the cortical extracts. Some results of quantitative analysis of cortical extracts are also reported, comparing different analytical procedures.

Triacylglycerol “hand-shape profile” of Argan oil. Rapid and simple UHPLC-PDA-ESI-TOF/MS and HPTLC methods to detect counterfeit Argan oil and Argan-oil-based products

The marketing of new argan-based products is greatly increased in the last few years and consequently, it has enhanced the number of control analysis aimed at detecting counterfeit products claiming argan oil as a major ingredient. Argan oil is produced in Morocco and it is quite expensive. Two simple methods for the rapid screening of pure oil and argan-oil based products, focused on the analysis of the triacylglycerol profile, have been developed. A three-minute-run by UHPLC-PDA allows the identification of a pure argan oil, while the same run with the MS detector allows also the analysis of products containing the oil down to 0.03%. On the other hand, by HPTLC the simultaneous analysis of twenty samples, containing argan oil down to 0.5%, can be carried out in a forty-five-minute run. The triglyceride profile of the most common vegetable fats such as almond, coconut, linseed, wheat germ, sunflower, peanut, olive, soybean, rapeseed, hemp oils as well as shea butter used either in cosmetics or commonly added for the counterfeiting of argan oil, has been also investigated. Over sixty products with different formulations and use have been successfully analyzed and argan oil in the 2.4-0.06% concentration range has been quantified. The methods are suitable either for a rapid screening or for quantifying argan oil in different formulations.