A fast isocratic liquid chromatography method for the quantification of xanthophylls and their stereoisomers.

A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13-Z-lutein, 13'-Z-lutein, 13-Z-zeaxanthin, all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, all-E-ß-apo-8'-carotenoic acid ethyl ester and all-E-ß-apo-8'-carotenal) within 12 min, compared to 90 min by the conventional high-performance liquid chromatography method. The separation was achieved on a YMC C30 reversed-phase column (100 mm x 2.0 mm; 3 µm) operated at 20°C using a methanol/tert-butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes.

Purification of Phenylalkanoids and monoterpene glycosides from Rhodiola rosea L. roots by high-speed counter-current chromatography.

INTRODUCTION: Rhodiola rosea L. is a medicinal herb used for its adaptogenic properties. The main active components are the phenylpropanoids collectively referred to as rosavins. OBJECTIVES: To develop an isolation method for phytochemicals present in Rhodiola rosea roots using high-speed counter-current chromatography (HSCCC). METHODOLOGY: The roots of Rhodiola rosea were extracted with methanol and fractionated using liquid-liquid partition and polyamide column clean-up. The purified fraction (100 mg) was subjected to semi-preparative HSCCC using the two-phase solvent system ethyl acetate:butanol:water (3:2:5). The head-to-tail elution mode was employed with a flow rate of 1.5 mL/min and a rotary speed of 1000 rpm. RESULTS: The separation yielded six main fractions with four components more than 90% pure. The sixth fraction was further purified using semi-preparative HPLC with a Synergi-hydro RP C18 -column to obtain rosin and geranyl 1-O-α-l-arabinopyranosyl(1 → 6)-ß-d-glucopyranoside. The main components isolated were rosavin (3.4 mg, 97% purity), salidroside (0.5 mg, 90% purity), benzyl-O-ß-d-glucopyranoside (1.2 mg, 85% purity), rosarin (1.3 mg, 99% purity), rosiridin (1.8 mg, 92% purity), rosin (1.2 mg, 95% purity) and geranyl 1-O-α-l-arabinopyranosyl(1 → 6)-ß-d-glucopyranoside (6.5 mg, 97% purity). The identity and purity of these components were confirmed using ultrafast liquid chromatography-diode-array detector-MS/MS analysis, ¹H- and ¹³C-NMR spectroscopy. CONCLUSION: High-speed counter-current chromatography was successful in the isolation of several phytochemicals present in Rhodiola rosea roots, including two components that are not commercially available.

Effect of domestic cooking methods on egg yolk xanthophylls.

Xanthophylls are a class of bioactive compounds known to play an important role in preventing age-related macular degeneration. Egg yolk is a rich source of highly bioavailable xanthophylls including lutein and zeaxanthin. The effects of domestic cooking methods (boiling, frying, microwaving) on egg yolk xanthophyll content were investigated. A LC-(APCI)-MS/MS method was used to identify and quantify all-E- and Z-isomers of lutein, zeaxanthin, canthaxanthin, and ß-apo-8'-carotenoic acid ethyl ester in fresh and cooked egg yolks. Both fresh and cooked yolks showed similar xanthophyll profiles but with higher contents of Z-isomers in cooked samples. All-E-lutein was the most affected, with 22.5%, 16.7%, and 19.3% reductions in boiled, microwaved, and fried yolk extracts, respectively. Total xanthophyll losses ranged from 6% to 18%. The results presented here could be useful in calculating the dietary intake of xanthophylls and also in assessing the xanthophyll profiles and contents of egg-containing products.

LC-MS/MS quantification of bioactive angiotensin I-converting enzyme inhibitory peptides in rye malt sourdoughs.

This study quantified antiotensin I-converting enzyme (ACE) inhibitory peptides in rye malt sourdoughs supplemented with gluten proteins and fermented with six strains of Lactobacillus spp. Bioinformatic analysis of prolamins from barley, rye, and wheat demonstrated that the ACE inhibitory peptides LQP, LLP, VPP, and IPP are frequently encrypted in their primary sequence. These tripeptides were quantified by liquid chromatography-tandem mass spectrometry. Tripeptide levels in sourdoughs were generally higher as compared to the chemically acidified controls. Sourdoughs fermented with different strains showed different concentrations of LQP and LLP. These differences corresponded to strain-specific differences in PepO and PepN activities. The highest levels of peptides VPP, IPP, LQP, and LLP, 0.23, 0.71, 1.09, and 0.09 mmol (kg DM)(-1), respectively, were observed in rye malt: gluten sourdoughs fermented with Lactobacillus reuteri TMW 1.106 and added protease. These concentrations were 6-7 times higher as compared to sourdough without fungal protease and exceed the IC(50) by 100-1000-fold.

Analysis of alkylamides in Echinacea plant materials and dietary supplements by ultrafast liquid chromatography with diode array and mass spectrometric detection.

Alkylamides are a class of compounds present in plants of the genus Echinacea (Asteraceae), which have been shown to have high bioavailability and immunomodulatory effects. Fast analysis to identify these components in a variety of products is essential to profile products used in clinical trials and for quality control of these products. A method based on ultrafast liquid chromatography (UFLC) coupled with diode array detection and electrospray ionization mass spectrometry was developed for the analysis of alkylamides from the roots of Echinacea angustifolia (DC.) Hell., Echinacea purpurea (L.) Moench, and commercial dietary supplements. A total of 24 alkylamides were identified by LC-MS. The analysis time for these components is 15 min. Compared to the alkylamide profiles determined in the Echinacea root materials, the commercial products showed a more complex profile due to the blending of root and aerial parts of E. purpurea. This versatile method allows for the identification of alkylamides in a variety of Echinacea products and presents the most extensive characterization of alkylamides in E. angustifolia roots so far.

Purification of alkylamides from Echinacea angustifolia (DC.) Hell. Roots by high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) was used for the separation of alkylamides from the roots of Echinacea angustifolia (DC.) Hell. For this purpose, the alkylamides were extracted with hexane and subjected to semipreparative HSCCC using a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (4:1:2:1). The lower aqueous phase was used as the mobile phase at a flow rate of 3 mL/min and a rotary speed of 1000 rpm. This procedure led to the isolation of four pure alkylamides, that is, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (38.9 mg, 97% purity), dodeca-2E,4E,8Z-trienoic acid isobutylamide (4.4 mg, 92% purity), dodeca-2E,4E-dienoic acid isobutylamide (3.2 mg, 99% purity), and dodeca-2E,4E-dienoic acid 2-methylbutylamide (0.3 mg, 92% purity). The identity and purity of the isolated alkylamides were confirmed by LC-ESI-MS and (1)H NMR and (13)C NMR data. To the best of the authors' knowledge, this is the first report of dodeca-2E,4E-dienoic acid 2-methylbutylamide in E. angustifolia roots.

Characterization and quantification of polyphenols in Amazon grape (Pourouma cecropiifolia Martius).

The phenolic profile of Amazon grape fruit (Pourouma cecropiifolia Martius) was investigated by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). For this purpose, suitable extraction and liquid chromatographic methods were developed. Anthocyanins, flavonols and chlorogenic acids were found mainly in the peel. Besides the main anthocyanins, i.e. delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-(6"-malonyl)glucoside, several minor anthocyanins were identified in the peel. Among these, cyanidin 3,5-diglucoside, delphinidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3-(3"-malonyl)glucoside, malvidin 3-glucoside, pelargonidin 3-glucoside, peonidin 3-glucoside and petunidin 3-glucoside were characterized on the basis of their fragmentation patterns in MS/MS experiments. The total anthocyanin content in the peel was 420.26±3.07 mg kg(-1) fresh weight. The pulp contained mainly 5-O-caffeoylquinic acid (210.39±3.43 mg kg(-1) fresh weight). Rutin was the predominant flavonol found in Amazon grape (peel 155.45 ± 2.06 mg kg(-1) fresh weight and pulp 2.64±1.21 mg kg(-1) fresh weight). Total polyphenols content was higher in the peel than in the pulp.

Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils.

The chemical composition of essential oils isolated from aerial parts of seven wild sages from Western Canada -Artemisia absinthium L., Artemisia biennis Willd., Artemisia cana Pursh, Artemisia dracunculus L., Artemisia frigida Willd., Artemisia longifolia Nutt. and Artemisia ludoviciana Nutt., was investigated by GC-MS. A total of 110 components were identified accounting for 71.0-98.8% of the oil composition. High contents of 1,8-cineole (21.5-27.6%) and camphor (15.9-37.3%) were found in Artemisia cana, A. frigida, A. longifolia and A. ludoviciana oils. The oil of A. ludoviciana was also characterized by a high content of oxygenated sesquiterpenes with a 5-ethenyltetrahydro-5-methyl-2-furanyl moiety, of which davanone (11.5%) was the main component identified. A. absinthium oil was characterized by high amounts of myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%). A. biennis yielded an oil rich in (Z)-beta-ocimene (34.7%), (E)-beta-farnesene (40.0%) and the acetylenes (11.0%) (Z)- and (E)-en-yn-dicycloethers. A. dracunculus oil contained predominantly phenylpropanoids such as methyl chavicol (16.2%) and methyl eugenol (35.8%). Artemisia oils had inhibitory effects on the growth of bacteria (Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis), yeasts (Candida albicans, Cryptococcus neoformans), dermatophytes (Trichophyton rubrum, Microsporum canis, and Microsporum gypseum), Fonsecaea pedrosoi and Aspergillus niger. A. biennis oil was the most active against dermatophytes, Cryptococcus neoformans, Fonsecaea pedrosoi and Aspergillus niger, and A. absinthium oil the most active against Staphylococcus strains. In addition, antioxidant (beta-carotene/linoleate model) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities were determined, and weak activities were found for these oils.