Article

Comparison of GC–MS and LC–MS methods for the analysis of antioxidant phenolic acids in herbs

Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland.
Analytical and Bioanalytical Chemistry (Impact Factor: 3.58). 07/2007; 388(4):881-7. DOI: 10.1007/s00216-007-1298-8
Source: PubMed

ABSTRACT Two methods were developed for the quantitative analysis of phenolic acids in herb extracts. The methods were based on liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) and gas chromatography-mass spectrometry (GC-MS). The methods were compared in terms of their linearity, repeatability, selectivity, sensitivity and the speed of the analysis. The sensitivity was good for both methods, with limits of detection of <80 ng/ml for most of the compounds. The relative standard deviations (RSD) of the peak areas were on average 7.2% for the LC-TOFMS method and 1.4% for the GC-MS method. Both methods were found to be suitable for the determination of the target analytes, although GC-MS was better suited to the quantitative determination of compounds present at low concentrations.

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    ABSTRACT: High dietary intake of fruits and vegetables has been linked to protection against chronic diseases, of which cardiovascular diseases (CVD) are the major cause of death in Finland. One of the hypotheses underlying the mechanism behind this effect is the antioxidant action of several dietary factors, including vitamins C and E, carotenoids and polyphenolic compounds. Polyphenols are consumed in large quantities in plant rich diets and observations on their antioxidant potency in vitro have made them a hot research topic during the past twenty years. The present study deals with the antioxidant activity of selected members of the plant family Lamiaceae sage (Salvia officinalis L.), thyme (Thymus vulgaris L.), rosemary (Rosmarinus officinalis L.) and two oregano species (Origanum vulgare L., Origanum onites L.). Due to the abundance of various phenolic compounds in these herbs, they are considered as a potential source of health-beneficial natural components with usage as health-promoting dietary supplements. The applicability of hydrodistillation, methanolic and ethanolic extraction, and pressurised hot-water extraction (PHWE) to extract phenolic antioxidants from sage was studied. Based on the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH ) scavenging activity of the different extracts, PHWE was the most effective technique. The hydrodistillation method was chosen for the further production of study extracts due to sensory properties of the extracts suitable for human consumption. The technique was also easily applicable in a larger scale production. The in vitro antioxidant properties of water extracts from sage, rosemary, thyme and oregano (O. vulgare) were characterised using three radical scavenging assays, a reductive capacity assay and a human low-density lipoprotein (LDL) oxidation assay. The total phenolic content of these extracts was determined. Depending on the method used, the extracts showed varying degrees of antioxidant activity. The absorption and metabolism of oregano (O. onites) extract in humans was evaluated by analysing the urinary excretion of phenolic metabolites using liquid chromatographic methods. The excretion of phenolic metabolites in urine was markedly increased following a single ingestion of O. onites extract, and the pattern of urinary metabolites suggested effective metabolism to take place. The in vitro antioxidant activity of phenolic constituents and human metabolites of O. onites were studied in DPPH and LDL oxidation assays. The phenolic acids from O. onites showed a range of antioxidant activity in the assays used, and some degree of activity was observed for many of the aglycone forms of their human metabolites. In a double-blinded, placebo-controlled clinical trial, the effects of oregano (O. vulgare) extract consumption on different biomarkers of antioxidant capacity and lipid peroxidation were studied in healthy men. No short- or long term effects on the antioxidant parameters followed were observed. This work revealed several aspects on the problematic nature of studies on antioxidants. Even though these extracts and their constituents seem promising antioxidant candidates in vitro and are at least to some extent available in the body, they lack effects on an array of lipid peroxidation and antioxidant capacity parameters in vivo. The findings suggest that their potential health-beneficial effects on humans are not mediated via direct antioxidant action. However, the epidemiologically observd association between the high intake of polyphenol rich fruits and vegetables and the decreased risk of CVD, as well as accumulating in vitro evidence on several bioactivities of polyphenols support the theory that polyphenols may have favourable effects on human health mediated via some other mechanisms. Epidemiologisten tutkimusten perusteella runsaasti kasviksia ja hedelmiä sisältävä ruokavalio vähentää riskiä sairastua sydän- ja verisuonisairauksiin. Yksi vahvimmista suojavaikutusta selittävistä teorioista pohjautuu ruokavalion hapettumista estävien eli antioksidatiivisten komponenttien, kuten C- ja E-vitamiinin, karotenoidien ja fenolisten yhdisteiden, toimintaan ihmiselimistössä. Tässä työssä tutkittiin Lamiaceae-heimon maustekasveja: salviaa (Salvia officinalis L.), timjamia (Thymus vulgaris L.), rosmariinia (Rosmarinus officinalis L.) ja kahta oreganolajiketta (Origanum vulgare L. ja Origanum onites L.)., jotka sisältävät runsaasti antioksidatiivisia fenolisia yhdisteitä. Työssä tutkittiin vesitislauksen, metanoli- ja etanoliuuton sekä paineistetun kuumavesiuuton käyttöä fenolisten antioksidanttien uuttamiseen salviasta. Uutteiden fenoliprofiili selvitettiin nestekromatografisesti ja niiden antioksidanttitehoa 1,1-difenyyli-2-pikryylihydratsyyliradikaalin (DPPH ) sieppaajina tutkittiin. Paineistettu kuumavesiuutto osoittautui menetelmistä tehokkaimmaksi. Osatöissä käytettävien uutteiden valmistamiseen valittiin vesitislaus, jolla valmistettujen uutteiden maku- ja hajuominaisuudet olivat käyttötarkoitukseen soveltuvat, ja joka oli helposti siirrettävissä suurempaan tuotantomittakaavaan. Salvian, rosmariinin, timjamin ja oreganon (O. vulgare) vesiuutteiden kokonaisfenolipitoisuus määritettiin, ja niiden antioksidanttiominaisuuksia koeputkimalleissa (in vitro) tutkittiin radikaalinsieppauskykyä, rautaionien pelkistämiskykyä ja low density lipoprotein (LDL) -kolesterolin hapettumisenestokykyä mittaavilla menetelmillä. Kaikki uutteet toimivat antioksidantteina käytetyissä koeputkimalleissa, mutta aktiivisuusjärjestys vaihteli menetelmittäin. Oreganon (O. onites) fenolisten happojen imeytymistä ja metaboliaa ihmiselimistössä arvioitiin tutkimalla uutteen nauttimisen jälkeen virtsaan erittyviä fenolisia metaboliitteja nestekromatografisesti. Metaboliittien määrä lisääntyi merkittävästi osoittaen, että ainakin osa uutteen fenolisista komponenteista imeytyi elimistöön. Yhdisteprofiilin perusteella uutteen fenoliset hapot metaboloituivat tehokkaasti. Saman uutteen fenolisten komponenttien ja niiden metaboliittien antioksidanttitehoa tutkittiin DPPH - ja LDL-malleissa. Uutteen fenolisilla yhdisteillä sekä niiden metaboliittien aglykonimuodoilla havaittiin antioksidanttiaktiivisuutta; metaboliittien aktiivisuudet olivat lähtöaineitaan alhaisempia. Kaksoissokkoutetussa, lumekontrolloidussa kliinisessä kokeessa tutkittiin, onko oreganouutteella (O. vulgare) vaikutuksia elimistön antioksidanttikapasiteettiin tai rasvojen hapettumista kuvastaviin mittareihin terveillä miehillä. Lyhyt- tai pitkäaikaisia vaikutuksia ei havaittu. Tutkitut yrtit sisälsivät voimakkaita vesiliukoisia antioksidantteja, joista suurin osa oli fenolisia happoja. Ainakin osa oreganon fenolisista yhdisteistä imeytyi ihmiselimistöön ja metaboloitui tehokkaasti. Siitä huolimatta 45 koehenkilöllä tehdyssä syöttökokeessa ei havaittu antioksidanttivaikutuksia yleisesti käytetyillä, luotettavina pidetyillä mittareilla. Tulokset viittaavat siihen, että yrttien fenolisten yhdisteiden mahdolliset terveyshyödyt eivät välittyisi suoran antioksidanttivaikutuksen kautta. Runsaasti fenolisia yhdisteitä sisältävän, kasvis-ja hedelmäpainotteisen ruokavalion epidemiologisesti havittu yhteys alentuneeseen sydän- ja verisuonisairauksien riskiin sekä fenolisten yhdisteiden lukuisat bioaktiiviset mekanismit koeputkimalleissa viittaavat kuitenkin siihen, että terveyshyötyjä voisi välittyä jonkin muun mekanismin kautta.
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