Guillermo Ramis-Ramos

Analysis of pharmaceutical preparations containing catecholamines by micellar liquid chromatography with spectrophotometric detection

An HPLC procedure for the determination of catecholamines (CAs) and other compounds which are associated with CAs in pharmaceutical preparations is described. A sodium dodecyl sulfate (SDS) micellar mobile phase, a C18 column and spectrophotometric detection at 280 nm were used. A mobile phase containing 0.1 mol l–1 SDS, 0.67 mol l–1(5% v/v) propanol and buffered with phosphate at pH 3 is recommended. The CAs gave limits of detection in the 4–7 ng ml–1 range. The procedure was applied to the determination of L-dopa, 2-methyldopa, epinephrine, dopamine and isoproterenol, as well as other compounds frequently associated with CAs in pharmaceuticals including phenylephrine, carbidopa and hydrochlorothiazide. The solute–micelle association constants and the partition constants of the CAs between the stationary phase and water in several media, and the protonation constants of L-dopa and 2-methyldopa, were evaluated from chromatographic data.

Use of triacylglycerol profiles established by high performance liquid chromatography with ultraviolet–visible detection to predict the botanical origin of vegetable oils

A method for the determination of triacylglycerols (TAGs) in vegetable oils from different botanical origins by HPLC with UV-vis detection has been developed. Using a core-shell particle packed column (C18, 2.6 μm), TAG separation was optimized in terms of mobile phase composition and column temperature. Using isocratic elution with acetonitrile/n-pentanol at 10 °C, excellent efficiency with good resolution between most of the TAG peak pairs, within a total analysis time of 15 min, was achieved. Using mass spectrometry detection, a total of 15 peaks, which were common to oils of six different botanical origins (corn, extra virgin olive, grapeseed, hazelnut, peanut and soybean) were identified. These peaks were used to construct linear discriminant analysis (LDA) models for botanical origin prediction. Ratios of the peak areas selected by pairs were used as predictors. All the oils were correctly classified with assignment probabilities higher than 95%.

Determination of cow's milk in non-bovine and mixed cheeses by capillary electrophoresis of whey proteins in acidic isoelectric buffers.

An improved method for the determination of cows milk in non-bovine cheese is reported: electrophoresis of whey proteins in acidic, isoelectric buffers. Two background electrolytes (BGEs) have been tested: (i) 50 mM iminodiacetic acid (pH=isoelectric point=2.30 at 25 degrees C), 0.5% hydroxyethylcellulose, 0.1% Tween 20 and 6 M urea (apparent pH 3.1), E=300 V/cm, for the separation of alpha-lactalbumins (alpha-LAs); (ii) a BGE with the same composition, but supplemented with 10% Tween 20, E=450 V/cm, for the fractionation of beta-lactoglobulins (beta-LGs). Surfactants have a discriminating effect on the retention behaviour of the bovine alpha-LA and beta-LG proteins, owing to the different strength of the protein-surfactant association complexes, and are needed for separating these two proteins from small peaks in the electropherograms generated by degradation of casein during cheese ripening. Novel equations are given for deriving the ratio of the area (or height) of bovine alpha-LA, or beta-LG, to the area (or height) of ovine or caprine alpha-LA or beta-LG (such ratios being typically used to determine the percentage of cows milk in dairy products), since previous equations had marked drawbacks, such as non-linearity of the plots with increasing slopes at high cows milk percentages, and too broad confidence limits at high cows milk contents, where the peak area (or height) ratio tends asymptotically to infinite. With the novel procedures reported, contents of cows milk as low as 1% can be quantified in goats and ewes cheeses. The present protocols give lower detection limits, are cheaper and more rapid than any other methodology reported in the literature, and can be easily applied to the routine quality control of binary and ternary cheeses.

Prediction of the genetic variety of Spanish extra virgin olive oils using fatty acid and phenolic compound profiles established by direct infusion mass spectrometry

The genetic varieties of Spanish extra virgin olive oils (Arbequina, Hojiblanca and Picual) were predicted by direct infusion of the samples in the electrospray ionization source of a mass spectrometer, followed by linear discriminant analysis of the spectral data. The samples were 1:50 diluted (v/v) with an 85:15 propanol/methanol (v/v) mixture containing 40mM KOH and infused. The abundances of the [M-H](-) peaks of the free fatty acids (7 peaks) and 28 phenolic compounds (20 peaks) were measured. Ratios of pairs of peak abundances were used as predictors in the construction of the linear discriminant analysis models. An excellent resolution between the three genetic varieties was achieved.

High-performance micellar liquid chromatography determination of sulphonamides in pharmaceuticals after azodye precolumn derivatization

A chromatographic procedure with precolumn derivatization to form the N-(1-naphthyl)ethylenediamine dihydrochloride azodyes is proposed for the analysis of several sulphonamides (sodium sulphacetamide, sulphadiazine, sulphaguanidine, sulphamerazine, sulphamethizole, sulphamethoxazole, sulphanilamide and sulphathiazole) in pharmaceutical preparations (tablets, pills, capsules, suspensions and drops). The separation is performed with a 0.05 M sodium dodecyl sulphate/2.4% pentanol eluent at pH 7. The precolumn derivatization improved the resolution in the chromatograms and increased the selectivity in the determination of mixtures of sulphonamides and in preparations where other drugs were present. The derivatization reaction was readily performed in a micellar medium of SDS at pH 1, leading to a rapid and simple procedure. The recoveries were in the 97-104% range with relative standard deviations usually below 3%.

Determination of cationic surfactants by capillary zone electrophoresis and micellar electrokinetic chromatography with deoxycholate micelles in the presence of large organic solvent concentrations

Mixtures of the cationic surfactants benzalkonium chloride (BKC) and cetylpyridinium chloride (CPC) were quickly resolved and reproducibly and reliably determined by using background electrolytes (BGEs) containing 80 mM borate, pH 8.5, bile salts and large concentrations of an organic solvent. When the bile salt is present, the separation mechanism changes from capillary zone electrophoresis (CZE) to a mixed micellar electrokinetic chromatography (MEKC)-CZE, with predominant MEKC interactions, which lead to an excellent resolution of all the solutes, including the C12-C18 homologues of BKC and CPC. A BGE containing 50 mM sodium deoxycholate and 30% ethanol for an extreme resolution, or 20% tetrahydrofuran for an adequate resolution within a much shorter analysis time, is recommended. The procedure was applied to the determination of the surfactants in industrial and household formulations, with excellent resolution between the homologues, detection limits of a few microg ml(-1) and reproducibilities below 2%.

γ-Oryzanol and tocopherol contents in residues of rice bran oil refining

Rice bran oil (RBO) contains significant amounts of the natural antioxidants γ-oryzanol and tocopherols, which are lost to a large degree during oil refining. This results in a number of industrial residues with high contents of these phytochemicals. With the aim of supporting the development of profitable industrial procedures for γ-oryzanol and tocopherol recovery, the contents of these phytochemicals in all the residues produced during RBO refining were evaluated. The samples included residues from the degumming, soap precipitation, bleaching earth filtering, dewaxing and deodorisation distillation steps. The highest phytochemical concentrations were found in the precipitated soap for γ-oryzanol (14.2 mg g(-1), representing 95.3% of total γ-oryzanol in crude RBO), and in the deodorisation distillate for tocopherols (576 mg 100 g(-1), representing 6.7% of total tocopherols in crude RBO). Therefore, among the residues of RBO processing, the deodorisation distillate was the best source of tocopherols. As the soap is further processed for the recovery of fatty acids, samples taken from every step of this secondary process, including hydrosoluble fraction, hydrolysed soap, distillation residue and purified fatty acid fraction, were also analyzed. The distillation residue left after fatty acid recovery from soap was found to be the best source of γ-oryzanol (43.1 mg g(-1), representing 11.5% of total γ-oryzanol in crude RBO).

Photo-polymerized lauryl methacrylate monolithic columns for CEC using lauroyl peroxide as initiator

Lauryl methacrylate (LMA)‐ester based monolithic columns photo‐polymerized using lauroyl peroxide (LPO) as initiator were prepared, and their morphological and CEC properties were studied. The composition of the polymerization mixture (i.e. ratios of monomers/porogenic solvents, 1,4‐butanediol/1‐propanol and LMA/crosslinker) was optimized. The morphological and chromatographic properties of LMA columns were evaluated by means of SEM pictures and van Deemter plots of PAHs, respectively. The polymerization mixture selected as optimal provided a fast separation of a mixture of PAHs with excellent efficiencies (minimum plate heights of 8.9–11.1 μm). Satisfactory column‐to‐column (RSD<4.5%) and batch‐to‐batch reproducibilities (RSD<6.3%) were achieved. The LMA columns photo‐polymerized with LPO were compared with those prepared with AIBN. Using PAHs, alkylbenzenes and basic compounds for testing, the columns obtained with LPO gave the best compromise between efficiency, resolution and analysis time.

RAPID LIQUID CHROMATOGRAPHIC DETERMINATION OF TETRACYCLINES IN ANIMAL FEEDS USING A SURFACTANT SOLUTION AS MOBILE PHASE

ABSTRACT A chromatographic procedure was developed for the determination of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) and minocycline (MINO) in animal feeds. Clear analyte-rich extracts were obtained using a 1 : 1 acetonitrile/water mixture buffered at pH 3. The extracts were injected into a conventional unprotected C18 chromatographic column and eluted with a mobile phase of 0.05 M sodium dodecyl sulfate/5% 1-butanol/0.01 M oxalic acid at pH 3. Good resolution was achieved for the five compounds, whereas OTC and TC coeluted with an optimized aqueous-organic mobile phase of methanol/acetonitrile/0.01 M oxalic acid at pH 3. Mean recoveries from spiked feed samples were 79, 83, 86, 87 and 95% for OTC, TC, CTC, DC and MINO, respectively, in the 15–100 µg g−1 concentration range. Limits of detection were in the range 0.1–0.4 µg mL−1. No interference was found from other antibiotics (penicillins G and V, amoxicillin, chloramfenicol, thiamfenicol, erythromycin and trimethoprim), sulfonamides (sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine and sulfathiazole), vitamin D3 and benzoic acid. The micellar system also permitted the resolution of CTC, OTC and TC from their 4-epimers. The methodology was applied to the analysis of animal feeds collected from cattle and pig growing farms.

Determination of cow's milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction

A novel method is reported for analyzing adulteration of goat and ewe cheeses with cows milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol‐water mixture in presence of 3 M urea and 1% β‐mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cows milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6—7%. In addition, the ripening time in goat and ewe cheeses could also be predicted.