A routine high-performance liquid chromatography method for carotenoid determination in ultrafrozen orange juices

An isocratic reversed-phase high-performance liquid chromatography method was developed for routine analysis of the main carotenoids related to the color of orange juice, using a more selective wavelength (486 nm) in which the absorption in the red-orange region of the visible spectra is maximum. Separation was carried out using as the mobile phase the mixture methanol:acetonitrile:methylene chloride:water (50:30:15:5, v/v/v/v), to which small amounts of butylated hydroxytoluene and triethylamine were added (0.1%). Identification was made by comparison either with standards obtained by thin-layer chromatography or with spectral data previously reported. The reproducibility of the method was remarkable; coefficients of variation for the most polar xanthophylls were under 1 and 4% for retention times and areas, respectively. Its application to Valencia late ultrafrozen orange juices has shown that major carotenoids are lutein + zeaxanthin (36%), lutein 5,6-epoxide (16%), antheraxanthin (14%), and beta-cryptoxanthin (12%).     

Characterization of paprika (Capsicum annuum) extract in orange juices by liquid chromatography of carotenoid profiles.

The carotenoid pigment profiles of authentic pure orange juices from Spain and Florida and an industrial paprika (Capsicum annuum) extract used for food coloring were obtained using reversed-phase liquid chromatography with a C18 packed column and an acetone/methanol/water eluent system. The procedure involving the carotenoid extraction is described. Both retention times and spectral properties using photodiode array detection for characterization of the major carotenoids at 430 and 519 nm are given. The influence of external addition of tangerine juice and/or paprika extract on orange juice color is described using the U.S. Department of Agriculture scale and adulterated orange juice. The procedure for quantitation of externally added paprika extract to orange juice is investigated, and the limit of quantitation, coefficient of variation, and recoveries are determined.     

Determination of carotenoid stereoisomers in commercial dietary supplements by high-performance liquid chromatography

A method for the determination of beta-carotene, lutein, and zeaxanthin including their cis-isomers and alpha-carotene in commercial dietary supplements by HPLC has been developed. The study comprises 11 oral dosage forms, including 9 soft gelatin capsules, 1 dragée, and 1 effervescent tablet formulation. The capsule content was extracted with an acetone-hexane mixture, and the gelatin shell was digested with papain to release carotenoids that had migrated into the coat. Sample preparation for tablets and dragées was carried out as described for the capsule content. Extraction recoveries exemplified for all-trans-beta-carotene and all-trans-lutein were 95 +/- 5% and 93 +/- 2%, and 95 +/- 2% and 79 +/- 5% after enzymatic treatment, respectively. Apart from all-trans-beta-carotene, its 9-cis- and 13-cis-isomers were detected in all samples, whereas no evidence for cis-isomerization of lutein and zeaxanthin could be obtained. Migration of carotenoids into the shells was only observed in the case of beta-carotene. With the exception of one preparation, the beta-carotene contents determined exceeded the dosage specified on the label by up to 48%, which results from stability overages necessary to compensate for losses during storage.     

Vineyard and fermentation studies to elucidate the origin of 1,8-cineole in Australian red wine

Preliminary investigations revealed that the proximity of Eucalyptus trees to grapevines can directly influence the concentration of the aroma compound 1,8-cineole present in the corresponding red wines. For two different vineyards, the closer the grapevines were to the trees, the greater was the amount of 1,8-cineole in the wines elaborated from those grapes. This led us to carry out further studies to quantify the levels of 1,8-cineole found in grape berries, leaves, and stems at set distances from Eucalyptus trees over multiple vintages. Generally, the highest concentration of 1,8-cineole was found in the grapevine leaves, followed by grape stems and then grapes. In each sample type, we observed greater concentrations of 1,8-cineole in samples closer to the trees. Various fermentation treatments carried out with Shiraz grapes showed that matter other than grapes (MOG, e.g., Eucalyptus or grape leaves) could contribute significant amounts of 1,8-cineole to the finished wines. These studies confirmed that vineyard position and winemaking conditions can determine the 1,8-cineole concentration in red wine. The fermentation study also showed for the first time that the concentration of rotundone in red wine can be strongly influenced by grapevine leaves and stems in the ferment.     

Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of anthocyanidins from berries and red wine is described. Delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin contents of bilberry (Vaccinium myrtillus), black currant (Ribes nigrum), strawberry (Fragaria ananassa cv. Jonsok), and a Cabernet sauvignon (Vitis vinifera) red wine were determined. The aglycon forms of the anthocyanins present in the samples were revealed by acid hydrolysis. A reversed phase analytical column was employed to separate the anthocyanidins before identification by diode array detection. The suitability of the method was tested by determining the recovery (95-102% as aglycons and 69-104% from glycosides) for each anthocyanidin. Method repeatability was tested by charting the total aglycon content of two samples over a period of 14 analyses and determining the coefficients of variation (1.41% for bilberry and 2.56% for in-house reference material). The method developed proved thus to be effective for reliable determination of anthocyanidins from freeze-dried berry samples and red wine. The total anthocyanidin content of the tested samples was as follows: in-house reference material, 447 +/- 8 mg/100 g; strawberry, 23.8 +/- 0.4 mg/100 g; black currant, 135 +/- 3 mg/100 g; bilberry, 360 +/- 3 mg/100 g; and Cabernet sauvignon red wine, 26.1 +/- 0.1 mg/100 mL.     

Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high pressure liquid chromatographic determination of retinyl palmitate and beta-carotene in oil and margarine.

A high pressure liquid chromatographic method was developed using high pressure gel permeation chromatography (HP-GPC) and high pressure reverse phase chromatography (RP-HPLC) for quantitation of retinyl palmitate and beta-carotene. HP-GPC was used for fractionation of vitamin A active compounds from oil preliminary to quantitation on nonaqueous RP-HPLC. HP-GPC fractionation was completed on oil and margarine dissolved in methylene chloride by 2 elution passes through 2 muStyragel (100 angstrom) columns connected in series with methylene chloride as the mobile phase. RP-HPLC separation of retinyl palmitate and beta-carotene was achieved on muBondapak C18 (10 micrometers), using methylene chloride-acetonitrile (30+70). Based on 10 repetitive analyses, recoveries of added beta-carotene and retinyl palmitate from vegetable oils were 98.6+/-2.9 and 95.2+/-2.6%, respectively. The coefficients of variation were 2.9% for beta-carotene and 2.7% for retinyl palmitate. The determination of vitamin A activity in 7 margarine brands with label claims of 10% U.S.RDA/serving revealed that all but one of the margarines contained at least 94% of the label claim. Vitamin A activity in the margarines ranged from 90.6 to 110.8% of the label declaration.     

Growth and essential oil composition responses of parsley cultivars to phosphorus fertilization and harvest date

Abstract

A field experiment was conducted on three cultivars (Italian Giant, Italian Plain, and Local) of parsley to compare plant growth, herb fresh weight, and essential oil (EO) content, yield and composition at three harvest times (Day 1, 47, and 91) in response to phosphorus (P) application rates (0, 12, 24, and 36?kg ha^?1). Repeated measures analysis revealed that the ideal P rate for the growth of parsley and its EO yield is 24?kg ha^?1; whereas the second harvest gives the highest height and weight; and the third harvest gives the highest EO content and yield. At all harvests, the highest EO content was obtained from Italian Giant fertilized with 36?kg ha^?1 P. β-myrcene, 1,3,8-p-menthatriene, β-phellandrene, and myristcin were the major compounds in all three cultivars, but their ideal P fertilization and harvest time varied with cultivar. This study showed biomass, EO content and yield, and the accumulation of EO constituents of parsley cultivars are influenced by P application and harvest date in different ways.     

Direct liquid chromatography method for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines

A validated HPLC method with fluorescence detection for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines is described. Detection conditions for both compounds were optimized (excitation at 279 and 278 and emission at 631 and 598 nm for hydroxytyrosol and tyrosol, respectively). The validation of the analytical method was based on selectivity, linearity, robustness, detection and quantification limits, repeatability, and recovery. The detection and quantification limits in red wines were set at 0.023 and 0.076 mg L(-1) for hydroxytyrosol and at 0.007 and 0.024 mg L(-1) for tyrosol determination, respectively. Precision values, both within-day and between-day (n = 5), remained below 3% for both compounds. In addition, a fractional factorial experimental design was developed to analyze the influence of six different conditions on analysis. The final optimized HPLC-fluorescence method allowed the analysis of 30 nonpretreated Spanish red wines to evaluate their hydroxytyrosol and tyrosol contents.     

Analysis of herbicides in olive oil by liquid chromatography time-of-flight mass spectrometry

The application of liquid chromatography time-of-flight mass spectrometry (LC/TOF-MS) for the identification and quantitation of four herbicides (simazine, atrazine, diuron, and terbuthylazine) in olive oil samples is reported here. The method includes a sample treatment step based on a preliminary liquid-liquid extraction followed by matrix solid-phase dispersion (MSPD) using aminopropyl as a sorbent material. A final cleanup step is performed with florisil using acetonitrile as an eluting solvent. The identification by LC/TOF-MS is accomplished with the accurate mass (and the subsequent generated empirical formula) of the protonated molecules [M + H]+, along with the accurate mass of the main fragment ion and the characteristic chlorine isotope cluster present in all of them. Accurate mass measurements are highly useful in this type of complex sample analyses since they allow us to achieve a high degree of specificity, often needed when other interferents are present in the matrix. The mass accuracy typically obtained is routinely better than 2 ppm. The sensitivity, linearity, precision, mass accuracy, and matrix effects are studied as well, illustrating the potential of this technique for routine quantitative analyses of herbicides in olive oil. Limits of detection (LODs) range from 1 to 5 microg/kg, which are far below the required maximum residue level (MRL) of 100 microg/kg for these herbicides in olive oil.     

Biological activity and chemical composition of the essential oil from Jamaican Hyptis verticillata Jacq

The chemical composition of the essential oil obtained by hydrodistillation from the aerial parts of Hyptis verticillata Jacq. was elucidated by a combination of GC and GC-MS analyses. The oil was dominated by the sesquiterpenoids cadina-4,10(15)-dien-3-one (15.1%) (1) and aromadendr-1(10)-en-9-one (squamulosone) (30.7%) (2). The oil exhibited chemosterilant activities against the cattle tick, Boophilus microplus Canest., and toxic action against adult Cylas formicarius elegantulus Summer, the most destructive pest of sweet potato (Ipomoea species).     

Separation of disaccharides by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Application to honey analysis

A new method based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToF MS) has been developed for the first time for the analysis of complex mixtures of disaccharides previously converted to their trimethylsilyl oximes (TMSO). Among the different experimental parameters considered for optimization, both the column set combination and the dimensions of the second-dimension column were found to be the most significant with regard to the complete resolution of structurally similar disaccharides. Application of the optimized method to honey analysis allowed the separation of most of the honey disaccharides previously described in the literature. Furthermore, 12 other unknown disaccharides have been separated by this method and characterized from their mass spectral data.     

Determination of the enantiomeric composition of gamma-lactones in edible oils by on-line coupled high performance liquid chromatography and gas chromatography

A new method is proposed for the determination of the enantiomeric composition of gamma-lactones in different vegetable edible oils (i. e., olive oil, almond oil, hazelnut oil, peanut oil, and walnut oil), and its potential for authenticity control is underlined for a limited number of samples. The method is based on the direct injection (i.e., without requiring a sample pretreatment step) in on-line coupled reversed phase liquid chromatography to gas chromatography (RPLC-GC) using a chiral stationary phase in the GC-step. Different experimental values for both speed of sample introduction into GC and volume of the transferred fraction are considered to improve the recoveries obtained. Relative standard deviations lower than 10% and detection limits ranging from 0.06 to 0.22 mg/L were achieved for the investigated gamma-lactones.     

Monoterpene composition of essential oil from peppermint (Mentha x piperita L.) with regard to leaf position using solid-phase microextraction and gas chromatography/mass spectrometry analysis.

Monoterpene compounds of leaf pairs and flowers of Mentha x piperita have been studied by direct headspace sampling using solid-phase microextraction coupled with gas chromatography/mass spectrometry (SPME-GC/MS). The content of peppermint-characteristic compounds such as menthol, menthyl acetate, and neomenthol increased in a basipetal direction (older plant parts), whereas menthone and isomenthone showed higher levels in the acropetal direction (younger plant parts). Higher levels of menthofuran were found in peppermint flowers in contrast to the leaves. SPME sampling resulted in relatively higher amounts of high-volatile monoterpenes and lower detection of less volatile compounds such as menthol and menthone, compared to solvent-based samples from essential oil distillation.     

Effects of rootstock on the composition of bergamot (Citrus bergamia Risso et Poiteau) essential oil

This paper reports the composition of bergamot oils obtained from plants grafted on the following rootstocks: sour orange, Carrizo citrange, trifoliate orange, Alemow, Volkamerian lemon, and Troyer citrange. The aim of this study is to evaluate the possibility of using rootstocks other than sour orange, checking their effect on the composition of the essential oil. Results are reported for analysis of 203 bergamot oils during the years 1997-1998, 1998-1999, and 1999-2000. The oils were analyzed by HRGC and HRGC/MS; 78 components were identified, and the results were in agreement with those reported in the literature for the Calabrian bergamot oils obtained from industry. Because of the quality of their essential oils, Alemow and Volkamerian lemon can be considered as substitutes for sour orange rootstocks.     

Chemical composition and antibacterial activity of the essential oil and the gum of Pistacia lentiscus Var. chia

The essential oil and gum of Pistacia lentiscus var. chia, commonly known as the mastic tree, are natural antimicrobial agents that have found extensive uses in medicine in recent years. In this work, the chemical composition of mastic oil and gum was studied by GC-MS, and the majority of their components was identified. alpha-Pinene, beta-myrcene, beta-pinene, limonene, and beta-caryophyllene were found to be the major components. The antibacterial activity of 12 components of mastic oil and the oil itself was evaluated using the disk diffusion method. Furthermore, attempts were made to separate the essential oil into different fractions in order to have a better picture of the components responsible for its antibacterial activity. Several trace components that appear to contribute significantly to the antibacterial activity of mastic oil have been identified: verbenone, alpha-terpineol, and linalool. The sensitivity to these compounds was different for different bacteria tested (Escherichia coli, Staphylococcus aureus, and Bacillus subtilis), which suggests that the antibacterial efficacy of mastic oil is due to a number of its components working synergistically. The establishment of a correlation between the antibacterial activity of mastic oil and its components was the main purpose of this research. Mastic gum was also examined, but it proved to be more difficult to handle compared to the essential oil.     

Application of denaturing high-performance liquid chromatography (DHPLC) for the identification of fish: a new way to determine the composition of processed food containing multiple species

The identification of fish species in transformed food products is difficult because the existing methods are not adapted to heat-processed products containing more than one species. Using a common to all vertebrates region of the cytochrome b gene, we have developed a denaturing high-performance liquid chromatography (DHPLC) fingerprinting method, which allowed us to identify most of the species in commercial crab sticks. Whole fish and fillets were used for the creation of a library of referent DHPLC profiles. Crab sticks generated complex DHPLC profiles in which the number of contained fish species can be estimated by the number of major fluorescence peaks. The identity of some of the species was predicted by comparison of the peaks with the referent profiles, and others were identified after collection of the peak fractions, reamplification, and sequencing. DHPLC appears to be a quick and efficient method to analyze the species composition of complex heat-processed fish products.     

Influence of plant growth stage on the essential oil content and composition in Davana (Artemisia pallens wall.)

The influence of three plant growth stages (full emergence of flower heads, anthesis, and initiation of seed set) on the essential oil content and composition in Davana (Artemisia pallens Wall) was investigated over two successive seasons. The essential oil content was found to be higher at the full emergence of flower heads than at anthesis and initiation of seed set stages. The contents of davanone, the major constituent of davana oil, and linalool decreased while those of (Z)- and (E)-methyl cinnamate, (E)-ethyl cinnamate, bicyclogermacrene, davana ether, 2-hydroxyisodavanone, and farnesol increased from flower heads emergence stage to the initiation of seed set stage. These results support the general practice of harvesting the crop at full bloom stage. Five compounds, viz., (Z)- and (E)-methyl cinnamates, (Z)- and (E)-ethyl cinnamates, and geranyl acetate, were identified for the first time in davana oil.     

Application of gas chromatography coupled to triple quadrupole mass spectrometry for the multiresidue analysis of pesticides in olive oil

A new multiresidue method has been developed and validated for the simultaneous determination of 100 pesticide residues in olive oil. The determination of pesticide residues was carried out in only 19 min by gas chromatography coupled to tandem mass spectrometry using a triple quadrupole mass analyzer. The mass spectrometer was operated in electron ionization and the selection reaction monitoring mode was used, acquiring two or three fragmentation reactions per compound. Two extraction processes were studied, and an evaluation of the stability and sensitivity of the chromatographic system has been performed for the tested extraction procedures. The final proposed methodology was based on a liquid-liquid partition with an n-hexane/acetonitrile mixture followed by a gel permeation chromatography cleanup step. An adequate lineal relation was obtained in the studied concentration range (10-200 microg kg (-1)); the recovery values were in the range 70-110% for the two levels of concentration studied: 12 and 50 microg kg (-1). Precision values, expressed as relative standard deviation, were lower than 18% at the aforementioned spiking levels; detection limits, confirmation limits, and quantitation limits were below or equal to 1.9, 2.6, and 3.6 microg kg (-1), respectively. The developed methodology was applied to the analysis of pesticide residues in real samples of olive oil from the south of Spain.     

Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states

The qualitative and quantitative composition of the essential oil from black, green, and white pepper was determined by using a simultaneous distillation and extraction micromethod for oil isolation and gas chromatography (GC)/flame ionization detection (FID) and GC/mass spectrometry (MS) analysis techniques. The most abundant compounds in pepper oils were (E)-beta-caryophyllene (1.4-70.4%), limonene (2.9-38.4%), beta-pinene (0.7-25.6%), Delta-3-carene (1.7-19.0%), sabinene (0-12.2%), alpha-pinene (0.3-10.4%), eugenol (0.1-41.0%), terpinen-4-ol (0-13.2%), hedycaryol (0-9.1%), beta-eudesmol (0-9.7%), and caryophyllene oxide (0.1-7.2%). Green pepper corn obtained by a sublimation drying method gave more oil (12.1 mg/g) and a much higher content of monoterpenes (84.2%) in the oil than air-dried green pepper corn (0.8 mg/g and 26.8%, respectively). The oil from ground black pepper contained more monoterpenes and less sesquiterprnes and oxygenated terpenoids as compared to green and white pepper oils. After 1 year of storage of pepper samples in a glass vessel at room temperature, the amount of the oils isolated decreased, the content of terpenes decreased, and the amount of oxygenated terpenoids increased. Differently from other pepper samples, 1 year storage of green pepper corn raised the oil amount more than twice of both drying methods.