Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

Monitoring aldehyde production during frying by reversed-phase liquid chromatography.

Acrolein (2-propenal) and other low molecular weight aldehydes (LMWAs) formed by degradation of the frying medium (triglycerides) were monitored by liquid chromatography (LC) during preparation of fried items. LMWA contents of coatings from codfish and of doughnuts and their volatiles that codistill with steam are monitored by trapping the vapors and distillate from the food matrix in a 2,4-dinitrophenylhydrazine solution. The resulting hydrazones are partitioned from the aqueous phase, first into isooctane and then into acetonitrile for LC analysis. The hydrazones are separated and quantified on a C18 reversed-phase column with acetonitrile-water as the mobile phase. LMWAs are confirmed by gas chromatography/mass spectrometry. No difference was found in LMWA content in coatings from fish fillets fried at 182 or 204 degrees C. Cake doughnuts were higher in acrolein content than yeast-raised doughnuts prepared under similar conditions. Freshness of the frying medium, frying time, and batch size did not seem to influence LMWA production from doughnuts. Results indicated that most of the LMWAs formed codistilled with steam during frying rather than remaining with the food item.     

Analysis of phenolic acids in barley by high-performance liquid chromatography

Phenolic acids from 30 barley varieties (combination of hulled/hulless/two-row/six-row/regular/waxy) were investigated by HPLC following four different sample treatments: (a) simple hot water extraction, (b) extraction after acid hydrolysis, (c) acid plus alpha-amylase hydrolysis, and (d) acid plus alpha-amylase plus cellulase hydrolysis treatments. The benzoic acid (p-hydroxybenzoic, vanillic, and protocatechuic acids) and cinnamic acid derivatives (coumaric, caffeic, ferulic, and chlorogenic acids) were identified, and some of the phenolic acids were quantified after each above-mentioned treatment. The data indicated that a combination of sequential acid, alpha-amylase, and cellulase hydrolysis treatments might be applicable for release of more phenolic acids from barley.     

Steviol quantification at the picomole level by high-performance liquid chromatography

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.     

Quantitative high-performance liquid chromatography analyses of flavonoids in Australian Eucalyptus honeys

Flavonoids of nine Australian monofloral Eucalyptus honeys have been analyzed and related to their botanical origins. The mean content of total flavonoids varied from 1.90 mg/100 g of honey for stringybark (E. globoidia) honey to 8.15 mg/100 g of honey for narrow-leaved ironbark (E. crebra) honey, suggesting that species-specific differences occur quantitatively among these Eucalyptus honeys. All of the honey samples analyzed in this study have a common flavonoid profile comprising tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone), which, together with myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and kaempferol (3,5,7,4'-tetrahydroxyflavone), were previously suggested as floral markers for European Eucalyptus honeys. Thus, flavonoid analysis could be used as an objective method for the authentication of the botanical origin of Eucalyptus honeys. Moreover, species-specific differences can also be found in the composition of honey flavonoid profiles. Among these honeys, bloodwood (E. intermedia) honey contains myricetin and tricetin as the main flavonoid compounds, whereas there is no myricetin detected in yapunyah (E. ochrophloia), narrow-leaved ironbark (E. crebra), and black box (E. largiflorens) honeys. Instead, these types of Eucalyptus honeys may contain tricetin, quercetin, and/or luteolin as their main flavonoid compounds. Compared to honeys from other geographical origins, the absence or minor presence of propolis-derived flavonoids such as pinobanksin, pinocembrin, and chrysin in Australian honeys is significant. In conclusion, these results demonstrate that a common flavonoid profile exists for all of the Eucalyptus honeys, regardless of their geographical origins; the individual species-specific floral types of Eucalyptus honey so common in Australia could be possibly differentiated by their flavonoid profile differences, either qualitatively or quantitatively or both.     

Measurement of food flavonoids by high-performance liquid chromatography: A review

The flavonoids are plant polyphenols found frequently in fruits, vegetables, and grains. Divided into several subclasses, they include the anthocyanidins, pigments chiefly responsible for the red and blue colors in fruits, fruit juices, wines, and flowers; the catechins, concentrated in tea; the flavanones and flavanone glycosides, found in citrus and honey; and the flavones, flavonols, and flavonol glycosides, found in tea, fruits, vegetables, and honey. Known for their hydrogen-donating antioxidant activity as well as their ability to complex divalent transition metal cations, flavonoids are propitious to human health. Computer-controlled high-performance liquid chromatography (HPLC) has become the analytical method of choice. Many systems have been developed for the detection and quantification of flavonoids across one, two, or three subclasses. A summary of the various HPLC and sample preparation methods that have been employed to quantify individual flavonoids within a subclass or across several subclasses are tabulated in this review.     

Separation of Delta5- and Delta7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.     

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).     

Separation and identification of nine penicillins by reverse phase liquid chromatography

A simple and rapid high performance liquid chromatographic method was developed for the separation and identification of amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, nafcillin, penicillin G potassium, and penicillin V potassium. The antibiotics were separated at ambient temperature on a Chromegabond 10 microns C18 column with acetonitrile-methanol-0. 01M potassium dihydrogen phosphate buffer, pH 4.7 (19 + 11 + 70), at 1 mL/min. A variable wavelength detector set at 225 nm, 0.16 AUFS , and a recorder set at 0.25 cm/min were used for the detection. Individual antibiotics and their mixtures were dissolved in the mobile phase and injected into the chromatograph through a 20 microL injection loop. Baseline separation was observed for virtually all 9 antibiotics. The entire mixture was resolved in less than 30 min. The method was sensitive, reproducible, and applicable to the qualitative analysis of commercial dosage forms.     

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.     

Rapid analysis of cholesterol-elevating compounds in coffee brews by off-line high-performance liquid chromatography/high-resolution gas chromatography

A new method is proposed to analyze the cholesterol-elevating cafestol and kahweol which allows their rapid and reliable determination in different coffee brews. The method involves the preseparation of the sample by high-performance liquid chromatography, the collection of the selected fraction, and its subsequent analysis by high-resolution gas chromatography using a programmed temperature vaporizer operated in the split mode as sampling system. Under the experimental conditions investigated, recoveries as high as 87% (cafestol) and 94% (kahweol) were achieved while detection limits equal to 0.06 and 0.04 ppm for cafestol and kahweol, respectively, were obtained. Examples are given comparing levels of cafestol and kahweol resulting from the same ground roasted coffee by different brewing methods, which show the lowest values for brews prepared from coffee bags.     

Purification of fusaproliferin from cultures of Fusarium subglutinans by preparative high-performance liquid chromatography

Thirty-six Fusarium strains were grown on cracked yellow corn and evaluated for optimum fusaproliferin production, with Fusarium subglutinans E-1583 producing the highest levels (1600 microg/g). Three solvent systems were tested for extracting fusaproliferin from the cultures of F. subglutinans E-1583. Methanol gave the highest fusaproliferin recovery, followed by methanol/1% aqueous NaCl (55:45, v/v) and acetonitrile/methanol/H(2)O (16:3:1, v/v/v). Hexane partitioning was effective in removing many impurities from the crude fusaproliferin extracts prior to the liquid chromatography step. Fusaproliferin samples were further purified by high-performance liquid chromatography (HPLC) with a C18 preparatory column using a mobile phase of acetonitrile/H(2)O (80:20, v/v). The purity of the fusaproliferin was verified by analytical HPLC, GC/MS, (1)H NMR spectroscopy, and electrospray ionization (ESI) MS. The isolated fusaproliferin was shown to be free of impurities and can be used as a standard for routine analysis. Fusaproliferin was shown to be temperature-sensitive when samples were stored at room temperature (20-24 degrees C) for more than several days. After 30 days at 4 degrees C, approximately 8% of the fusaproliferin had been transformed to deacetyl-fusaproliferin; however, samples stored at -20 degrees C for 1 year contained only trace amounts of the deacetylated form.     

Improved normal-phase high-performance liquid chromatography procedure for the determination of carotenoids in cereals

Besides the health benefits associated with whole-grain consumption, cereals are recognized sources of health-enhancing bioactive components such as carotenoids, which are a group of yellow pigments involved in the prevention of many degenerative diseases and which have been used for a long time as indicators of the color quality of durum wheat and pasta products. This work reports a fast, sensitive, and selective procedure for the extraction and determination of carotenoids from cereals and cereal byproducts. The method involves sample saponification and extraction followed by normal-phase high-performance liquid chromatography, allowing the separation of the main carotenoids pigments of cereals, especially lutein and zeaxanthin. An application of the established method to various species of cereals and cereal byproducts is also shown. The highest carotenoid levels were found in maize (approximately 11.14 mg/kg of dry weight), which contains high amounts of beta-cryptoxanthin (2.40 mg/kg of dry weight), and, among the cereals considered, has the highest content of zeaxanthin (6.43 mg/kg of dry weight) and alpha+beta-carotene (1.44 mg/kg of dry weight). With the exception of maize, lutein is the main compound found (from 0.23 to 2.65 mg/kg of dry weight in oat and durum wheat, respectively). Moreover, whereas alpha+beta-carotene and zeaxanthin are principally localized in the germ, lutein is equally distributed along the kernel.     

Analysis of eight capsaicinoids in peppers and pepper-containing foods by high-performance liquid chromatography and liquid chromatography-mass spectrometry

Diverse procedures have been reported for the isolation and analysis of secondary metabolites called capsaicinoids, pungent compounds in the fruit of the Capsicum (Solanaceae) plant. To further improve the usefulness of high-performance liquid chromatography (HPLC), studies were carried out on the analysis of extracts containing up to eight of the following capsaicinoids: capsaicin, dihydrocapsaicin, homocapsaicin-I, homocapsaicin-II, homodihydrocapsaicin-I, homodihydrocapsaicin-II, nonivamide, and nordihydrocapsaicin. HPLC was optimized by defining effects on retention times of (a) the composition of the mobile phase (acetonitrile/0.5% formic acid in H2O), (b) the length of the Inertsil column, and (c) the capacity values (k) of the column packing. Identification was based on retention times and mass spectra of individual peaks. Quantification was based on the UV response at 280 nm in HPLC and recoveries from spiked samples. The method (limit of detection of approximately 15-30 ng) was successfully used to quantify capsaicinoid levels of parts of the pepper fruit (pericarp, placenta, seeds, and in the top, middle, and base parts of whole peppers) in 17 species of peppers and in 23 pepper-containing foods. The results demonstrate the usefulness of the method for the analysis of capsaicinoids ranging from approximately 0.5 to 3600 microg of capsaicin equiv/g of product. The water content of 12 fresh peppers ranged from 80.8 to 92.7%. The described freeze-drying, extraction, and analysis methods should be useful for assessing the distribution of capsaicinoids in the foods and in defining the roles of these biologically active compounds in the plant, the diet, and medicine.     

Determination of bisphenol a and bisphenol B residues in canned peeled tomatoes by reversed-phase liquid chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.     

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%).     

Simultaneous assay for six alkaloids in opium, using high-performance liquid chromatography.

A method is described for the quantitative determination of morphine, codeine, cryptopine, thebaine, papaverine, and narcotine in opium by high-performance liquid chromatography. The alkaloids are isolated from a dilute acid extract by adsorption on an Amberlite XAD-2 resin column and eluted first with methanol and then with chloroform-methanol (3+1). After solvent removal by reduced pressure evaporation, the alkaloids are redissolved in chloroform-methanol (3+1). The sample solution, plus brucine as an internal standard, is injected onto a Corasil II column and eluted with hexane that is gradient programmed with a solution of chloroform-methanol-diethylamine (100+300+1). The absorbances of the separated alkaloids are continuously monitored at 254 nm, using a flow-through ultraviolet double-beam photometer.     

Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC)

Polyphenolic compounds of apple may play an important role in physiologic functions related to human health. Different polyphenolics may have varied biological activities including antioxidant activity. The objective of this study was to investigate the profiles of polyphenolic compounds in different apple varieties and different parts of an apple. The total and individual polyphenolics differed significantly among the eight apple cultivars grown in Ontario, and the peels had higher concentrations than the flesh. Among the tested cultivars, Red Delicious and Northern Spy had the highest concentrations and Empire the lowest. Five major polyphenolic groups with a total of 16 identified individual compounds were found, among which the dihydroxycinnamic acid esters, phloretin glycosides, and flavan-3-ols were found in both flesh and peel, whereas quercetin glycosides were almost exclusively found in the peel. Cyanidin 3-galactoside was unique to and found only in red apple peels. In both apple peel and flesh, the predominant group of polyphenolics was the procyanidins, followed by quercetin glycosides in the peel and hydroxycinnamic acid esters in the flesh. 3-Hydroxyphloretin 2'-xyloglucoside was newly identified in apple. The results obtained in this study will further the understanding of the polyphenolic composition of apples and their roles in health-promoting physiological functions.     

Saponins composition in American ginseng leaf and berry assayed by high-performance liquid chromatography

The root of American ginseng is a commonly used herbal medicine in the United States. However, the compositions of American ginseng leaves and berries are not clear to date. In this study, we improved a method for the analysis of 12 ginsenosides based on solid phase extraction and high-performance liquid chromatography-ultraviolet. Good resolution was obtained for all tested ginsenosides: Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, 20(R)-Rg2, Rg3, Rh1, and Rh2. Ginsenosides Rh1, Rg2, and 20(R)-Rg2 were easily separated with this column. The modified gradient elution program resulted in satisfactory linearity and precision. Solid phase extraction made the analysis accurate and efficient. Other investigators recently observed that ginsenoside Rb3 is a potent neuroprotective compound; it can promote learning and memory. In this report, we found that the major ginsenoside in American ginseng leaves and berries was ginsenoside Rb3, while Rb3 only had limited amounts in the root of American ginseng and other species of the Panax genus. Ginsenoside Rb3 was quantified as 4.71% in American ginseng leaves and 5.35% in berries, suggesting that American ginseng leaves and berries are new sources of ginsenoside Rb3.     

Determination of major carotenoids in a few Indian leafy vegetables by high-performance liquid chromatography

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.