Development and validation of a HPLC method for the bioactive composition characterization of plant materials

Abstract

Tekućinska kromatografija visoke djelotvornosti (HPLC) danas predstavlja nezaobilazan korak u analizi bioaktivnog sastava biljnih supstrata. Međutim, postoji potreba za razvojem jedinstvene i univerzalne metode koja bi omogućila, brzu, rutinsku i pouzdanu analizu velikog broja strukturno različitih bioaktivnih spojeva, iz botanički raznovrsnih uzoraka, kao što su ekstrakti ljekovitih biljnih vrsta. Stoga je cilj ovog rada bio razviti jedinstvenu HPLC-DAD metodu za određivanje polifenolnih spojeva u određenim ljekovitim biljnim vrstama. Osim razvoja i validacije HPLC-DAD metode provedena je spektrofotometrijska analiza polifenolnog sastava vodenih ekstrakata šesnaest biljnih vrsta. Najveći udjel ukupnih polifenola, hidroksicimetnih kiselina, flavona i flavonoida te najveći antioksidacijski kapacitet određeni su u ekstraktima biljnih vrsta iz porodice Lamiaceae (paprena metvica, matičnjak). Vrijednosti granice detekcije i kvantifikacije te visoka vrijednost točnosti i preciznosti pokazuju prikladnost razvijene HPLC metode za kvantifikaciju polifenolnih spojeva u ispitivanim biljnim vrstama. Novorazvijenom HPLC metodom u ispitivanim biljnim vrstama određeno je ukupno 24 spoja koji pripadaju hidroksibenzojevim i hidroksicimetnim kiselinama te flavonoidima. Primjenom razvijene metode, određen je karakterističan visok udjel ružmarinske kiseline te luteolina u biljkama iz porodice Lamiaceae, te pojedinačni polifenolni spojevi identificirani u samo jednoj biljnoj vrsti što se može smatrati njihovim svojevrsnim taksonomskim biomarkerima.Nowadays, high performance liquid chromatography (HPLC) represents an unavoidable step in the analysis of bioactive composition of plant substrates. However, there is a need for the development of a unique and universal method which would enable rapid, reliable and routine analysis of a large number of structurally diverse bioactive compounds from botanically different samples, such as medicinal plant extracts. Therefore, the aim of this study was to develop a unique HPLC-DAD method for determination of polyphenolic compounds in certain medicinal plants. In addition to the development and validation of HPLC-DAD method, spectrophotometric analysis of polyphenolic compound from aqueous extracts of sixteen medicinal plant species was carried out. The highest content of total polyphenols, hydroxycinnamic acids, flavones and flavonoids and the most potent antioxidant capacity were determined in extracts of the Lamiaceae plants (peppermint, lemon balm). The values of the limit of detection and quantification and high values determined for the accuracy and precision show the suitability of the developed HPLC method for the quantification of polyphenolic compounds in the evaluated plant species. The newly developed HPLC method in the studied plant species enabled the separation and determination of 24 polyphenolic compounds belonging to the hydroxybenzoic and hydroxycinnamic acids and flavonoids. By employing the developed method, high contents of rosmarinic acid and luteolin, characteristic for the plants of Lamiaceae family were determined, as well as individual polyphenolic compounds identified in only one plant species, which can be considered as a kind of their taxonomic biomarkers