Fabio Zonta

High-performance liquid chromatographic determination of carotene and vitamin A and its geometric isomers in foods : Applications of cheese analysis

Abstract A fractionation of retinol geometric isomers and of carotene was achieved, by straight-phase high-performance liquid chromatography, using a LiChrosorb Si 60 5- μm prepacke column and an isocratic mobile phase of methyl ethyl ketone—hexane (10:90). The wavelengths selected for detection of caroten and retinols were 450 nm and 340 nm respectively, and wre changed during the chromatographic run. Two internal standards were used for quantitative analysis: 2-nitrofluorene for retinols and azobenzene for carotene. The proposed method, which has the advantage that all vitamin A active compounds can be evaluated simultaneously, was applied t the analysis of Italian cheese samples. The percentage recoveries of retinol and carotene were 75.7 and 79.6, respectively, and were not affected by cheese fat content, at least in the explored range.

High-performance liquid chromatography of the unsaponifiable from samples of marine and freshwater fish: fractionation and identification of retinol (vitamin A1) and dehydroretinol (vitamin A2) isomers

Retinol (vitamin A1) and dehydroretinol (vitamin A2) geometric isomers, obtained in photosynthetic mixtures or from natural samples, were simultaneously fractionated by means of isocratic normal phase high-performance liquid chromatography. The use of low percentages of isopropanol (0.4-1.1%) as a modifier in hexane was studied, and the resolution of both pairs of retinols (9-cis/all-trans and 13-cis/11-cis) whose baseline separation had not previously been achieved was obtained. The proposed chromatographic method therefore allows the complete separation of all six of the most commonly occurring retinols in natural samples, including the above four mono cis- retinols plus the two 9,13- and 11,13-di-cis- retinols . Photoisomerization of retinal and of retinol in different solvent systems and during the extraction process was also studied, before analyzing the unsaponifiable from fresh and marine water fish, which constitute rich natural sources of both vitamins A1 and A2. Vitamin A2 was found to be present in different relative percentages depending on the analytical matrix. Four cis isomers (9-cis-, 11-cis-, 13-cis- and 9,13-di-cis-retinol or corresponding dehydroretinols ) were found to occur naturally together with the main all-trans form, confirming the need to separate geometric isomers in every dosage of vitamin A-containing compounds.

High-performance liquid chromatography of fat-soluble vitamins : Separation and identification of vitamins D2 and D3 and their isomers in food samples in the presence of vitamin A, Vitamin E and carotene

Vitamins D2 and D3 and their corresponding previtamins and provitamins were resolved by reversed-phase high-performance liquid chromatography using a ternary solvent system (acetonitrile-methanol-water) pumped according to a gradient elution programme. The D vitamins were also resolved in the presence of other lipid-soluble vitamins (A, E and K1) and carotene. The peaks were monitored with a UV-visible variable-wavelength detector and were detected at their maximum absorbance, resulting in maximum sensitivity. Lipid-soluble vitamins and carotene were resolved in extracts obtained from oils and butter, thus permitting their identification in a single chromatographic run.

High-performance liquid chromatography of fat-soluble vitamins. Simultaneous quantitative analysis of vitamins D2, D3 and E. Study of percentage recoveries of vitamins from cod liver oil.

Vitamins D2, D3 and E were resolved and quantified by applying reversed-phase high-performance liquid chromatography to extracts of cod liver oil. The method, using two reversed-phase C18 columns and a ternary mixture of acetonitrile, methanol and water as the eluent resolved all fat-soluble vitamins well, including the pair D2-D3. The extraction procedure was studied; the recoveries, using two different solvents (hexane and diethyl ether) for extractions were 60.6 +/- 1.0 and 77.1 +/- 1.1, 56.9 +/- 1.2 and 74.8 +/- 0.8, and 14.1 +/- 0.7 and 89.8 +/- 1.4% for vitamins D2, D3 and E, respectively.

High-performance liquid chromatography of retinals, retinols (vitamin A1) and their dehydro homologues (vitamin A2): improvements in resolution and spectroscopic characterization of the stereoisomers

A study of mobile phases for the improved high-performance liquid chromatographic resolution of retinal, 3-dehydroretinal, retinol and 3-dehydroretinol stereoisomers is described. By using 1-octanol as a phase modifier in n-hexane, the simultaneous separation of a complex mixture of vitamin A1 and A2 isomers was satisfactorily achieved, while the separation of the corresponding aldehydes required a ternary mixture of 2-propanol, dioxane and n-hexane. All peaks were spectroscopically characterized by recording their UV spectra, which are reported together with comparative tables of the absorbance maxima of the stereoisomers.

Simultaneous high-performance liquid chromatographic analysis of free carotenoids and carotenoid esters.

High-performance liquid chromatography using a non-aqueous reversed phase with gradient elution on C18 columns is a powerful tool for investigating the carotenoid composition of natural samples, e.g., flower petals, and for the simultaneous detection of carotenoids of the widest possible polarity range (xanthophylls, diones, hydrocarbons and carotenoid esters). The comparison of sample extracts submitted or not to saponification allows the presence of carotenoid esters to be revealed through the appearance of the corresponding free hydroxycarotenoids. The gas chromatographic analysis of the fatty acids after alkaline hydrolysis of esters provides further confirmation. In most cases, peaks of various carotenoids were identified by comparison with standards. The wavelengths of the visible absorbance maxima of the chromatographed carotenoids as obtained on-line by the stop-flow method in the eluent system and off-line in carbon disulphide are reported. The esters appear to constitute the main carotenoid fraction in flower petals.

Quantitative high-performance liquid chroamtographic method for determining the isomer distribution of retinol (vitamin A1) and 3-dehydroretinol (vitamin A2) in fish oils

Seven retinol and seven 3-dehydroretinol geometric isomers were simultaneously fractionated on a silica column using a ternary mobile phase (2-propanol-1-octanol-n-hexane, 0.2:3.8:96). The method was applied to determine vitamin A active compounds present in fish oils. The standard additions method was used to obtain the recovery of the analytes, which was found to be the same for vitamin A1 and A2 (89%). Correction factors for determining all isomers when the peak areas are integrated at 326 nm are reported, so that the quantitative determination may be reproduced by others using the proposed method, which allows all data to be referred to the calibration graph of all-trans-retinol only. As every isomer is quantified separately, the method also permits the determination of the real vitamin A biopotency of the samples.

Quantitative analysis of phylloquinone (vitamin k1) in soy bean oils by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining phylloquinone (vitamin K1) in soy bean oils is described. Resolution of vitamin K1 from interfering peaks of the matrix was obtained after enzymatic digestion, extraction and liquid-solid chromatography on alumina. An isocratic reversed-phase chromatography with UV detection was used in the final stage. The quantitation was carried out by the standard addition method, and the recovery of the whole procedure was 88.2%.

High-performance liquid chromatography of carotenoids from some marine shellfish

The chromatographic profiles of carotenoids from some marine shellfish are reported. The analyses were performed both by gradient elution reversed-phase and isocratic direct phase high-performance liquid chromatography (HPLC), and the results obtained are compared and discussed. The six main carotenoids extracted from three shellfish species (Chlamys opercularis, L., Cardium tuberculatum, L., Pecten jacobaeus, L.) were isolated in highly pure form by means of semi-preparative HPLC. They were characterized by means of spectroscopic methods (visible absorption and infrared spectroscopy), and chemical tests: the identification of fucoxanthin and alloxanthin was possible while a tentative identification of two other carotenoids (fucoxanthinol and diatoxanthin) was suggested. Carotenoids in clams and scallops were present both in esterified and free form, while in cardia only carotenoid esters were detected.