Chemical analysis of saffron by HPLC based crocetin estimation.

Saffron is one of the most expensive and valuable spice having tremendous commercial value in food industry and thus its quality check is of utmost importance. Crocins are unique group of extremely hydrophilic apocarotenoids which are main components of saffron. Crocetin is an aglycone of crocins which do occur naturally in saffron, and is produced in biological system as a bioactive metabolite via hydrolytic cleavage of crocins. Crocins are unstable and tend to undergo isomerisation/ inter-conversions, and therefore their quantitative estimation is difficult. Herein, we have established for the first time, a crocetin-based HPLC method to evaluate the total crocin content of saffron, and thereby analyze the quality of saffron. The present approach comprises alkali-mediated conversion of crocins to crocetin in raw material followed by quantitative estimation of in-situ formed crocetin by HPLC analysis. The unique and efficient protocol for preparation of high purity analytical grade 'crocetin' directly from saffron has also been established. It is simple and efficient way to check the quality of saffron/ saffron-containing products.

Ultra high performance liquid chromatography versus high performance liquid chromatography: stationary phase selectivity for generic carotenoid screening.

Aim of study was to find the most suitable LC column for generic carotenoid screening. To represent the diversity of carotenoids in nature and to optimize chromatographic separation, a set of carotenoid standards was carefully chosen to account for the various classes of carotenoids. The HPLC C30 column has since long been the 'golden standard' in the chromatographic separation of carotenoids. Since approximately one decade, new UHPLC technology has led to much shorter analysis times, smaller peak widths and higher chromatographic resolution. However, there are currently no UHPLC columns on the market containing the specific stationary phase chemistry of the HPLC C30 column. Therefore during this study, we investigated the separation of carotenoids on a set of UHPLC columns and compared it to their separation on the HPLC C30 column. Comparison of carotenoids separations on the different stationary phases with objective column comparison parameters clearly indicated that the HPLC C30 column is an overall better performer in the separation of carotenoids. This is due to the lack of UHPLC column chemistries that are adapted for carotenoid analysis. However, analysis time on the HPLC C30 column takes about four times longer compared to UHPLC analysis. Therefore, with the range of columns that are commercially available nowadays, a choice has to be made between very high selectivity (HPLC C30 column) and analysis times that are adapted to modern laboratory requirements (UHPLC technology). Therefore, carotenoid separations would be even more performing if an appropriate UHPLC C30 column would be available.

Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

Evaluation of certain food additives and contaminants.

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, with a view to recommending acceptable daily intakes (ADIs) and to preparing specifications for identity and purity. The Committee also evaluated the risk posed by two food contaminants, with the aim of deriving tolerable intakes where appropriate and advising on risk management options for the purpose of public health protection. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of and assessment of dietary exposure to food additives and contaminants. A summary follows of the Committee's evaluations of technical, toxicological and dietary exposure data for certain food additives (aluminium-containing food additives, Benzoe Tonkinensis, glycerol ester of gum rosin, glycerol ester of tall oil rosin, glycerol ester of wood rosin, octenyl succinic acid modified gum arabic, polydimethyl siloxane, Ponceau 4R, pullulan, pullulanase from Bacillus deromificans expressed in Bacillus licheniformis, Quinoline Yellow and Sunset Yellow FCF) and two food contaminants (cyanogenic glycosides and fumonisins). Specifications for the following food additives were revised: aluminium lakes of colouring matters; beta-apo-8'-carotenal; beta-apo-8'-carotenoic acid ethyl ester; beta-carotene, synthetic; hydroxypropyl methyl cellulose; magnesium silicate, synthetic; modified starches; nitrous oxide; sodium carboxymethyl cellulose; and sucrose monoesters of lauric, palmitic or stearic acid. Annexed to the report are tables summarizing the Committee's recommendations for dietary exposures to and toxicological evaluations of the food additives and contaminants considered.

A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study.

Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, beta-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.

Use of non-aqueous capillary electrophoresis for the quality control of commercial saffron samples.

A non-aqueous capillary electrophoresis (NACE) method for quantifying the seven crocin metabolites that are the major biologically active ingredients of saffron was developed. Separation is done by using a fused silica capillary filled with a 12.5 mM H3BO3/37.5 mM sodium tetraborate methanolic solution as background electrolyte. The results obtained were compared with the total index "safranal value", widely used as a quality measure of saffron products. The comparison revealed that the proposed NACE method provides useful information not obtained in the safranal value. Infact, samples with a similar safranal value can contain crocin metabolites in different concentrations and relative proportions. This new method is very useful for quality control in commercial saffron samples.

A simplified method for the HPLC resolution of alpha-carotene and beta-carotene (trans and cis) isomers.

A new HPLC/DAD (Diode Array Detector) method is proposed for the identification of some carotene isomers. The operating conditions adopted permit the resolution of alpha-carotene, all-trans-beta-carotene, 9-cis-beta-carotene, 13-cis-beta-carotene and 15-cis-beta-carotene. Moreover, the chromatographic conditions reported are simplified in respect of those reported up to now. The method is applied to the determination of carotenoids in a dried Dunaliella salina extract, but it could be also applied to other organic matrices such as eggs.

Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector.

Numerous epidemiological studies have linked carotenoids to cancer preventive processes, thereby increasing interest in levels of these micronutrients in human tissue and serum. Conventional analyses of these biological tissues employ liquid chromatography (LC) with ultraviolet and visible absorbance (UV-VIS) detection. However, this type of carotenoid analysis does not provide adequate sensitivity for very small sample sizes, such as microscale biopsies, when only small quantities of tissue are available. Electrochemical detection (ECD) is a useful alternative to conventional UV-VIS detection methods for LC analysis of carotenoids in cases where high sensitivity is necessary. Both hydrocarbon (beta-carotene and alpha-carotene) and oxygenated carotenoids (lutein, zeaxanthin, and beta-cryptoxanthin) were detected at electrical potential settings between 220 and 520 mV. The generated electrochemical array data (hydrodynamic voltammograms) can be used to identify carotenoids as well as to differentiate between trans and cis configurations. Detection limits for beta-carotene by ECD were measured at 10 fmol representing approximately a 100- to 1000-fold increase over conventional LC-UV-VIS techniques. The developed methodology was applied successfully to microscale analysis of biological tissues.

In vivo cytogenetic studies on male mice exposed to Ponceau 4R and beta-carotene.

Chromosomal aberrations induced by Ponceau 4R (an azo food dye) and beta-carotene (a natural food colour) were studied on bone marrow cells of mice in vivo. The results indicated that Ponceau 4R was more clastogenic than beta-carotene. Ponceau 4R was found to have a minimum effective dose of 4 mg which induced a significant number of chromosome aberrations. This dose is also the recommended 'admissible daily intake'. Chromosome aberrations induced by beta-carotene were not significantly higher than those of the control (olive oil) in the dose range 0.27 to 27 mg/kg body weight. The genotoxicity of these dyes can be attributed to their chemical composition. In so far as genotoxicity is concerned the carotenoid beta-carotene can be safely used as a food colorant whereas Ponceau 4R should be delisted as a food dye.

The safety of beta-carotene.

Epidemiological studies have associated low dietary and/or plasma level of carotenoids with higher incidences of certain cancers. This evidence has led the National Cancer Institute to initiate more than a dozen prospective clinical trials in which supplements of beta-carotene alone, or in combination with other micronutrients, are being taken. In these trials, the beta-carotene supplements are given in the range of 15-50 mg/day. The safety of this level of intake is well documented. beta-Carotene has been successfully used to treat inherited photosensitivity diseases for more than 15 years at dosages of 180 mg/day or more, without any adverse effects other than hypercarotenemia. Toxicity studies in animals have shown that beta-carotene is not carcinogenic, mutagenic, embryotoxic, or teratogenic and does not cause hypervitaminosis A. In the few isolated reports of carotenoid-related toxicity, the findings are associated with very large intakes of foods containing beta-carotene, among other constituents, and have not been substantiated in individuals who have taken high doses of beta-carotene for several years.

A specific method for the determination of provitamin A carotenoids in orange juice.

A method has been developed for rapidly determining the amounts of alpha-carotene, beta-carotene, and cryptoxanthin in orange juice. The procedure includes extraction, saponification, and high-speed liquid chromatography. Limits of detection for the 3 carotenoids are 0.04, 0.02, and 0.04 mug/ml, respectively.