Hesperidin, eriocitrin, rutin ter kavna in klorogena kislina so antimikrobne snovi, ki jih najdemo v limoni in njenem suhem ekstraktu. Zaradi njihovih pozitivnih učinkov, predvsem na zdravje ljudi, se vse bolj pogosto uporabljajo v raznih prehrambenih dodatkih. V magistrskem delu smo razvili in optimizirali HPLC metodo, s katero identificiramo in kvantificiramo te antimikrobne snovi v suhem ekstraktu limone.
Za ločevanje spojin smo uporabili gradientno elucijo, kjer smo 0,4 % ocetni kislini postopoma dodajali vse več organske faze (metanola) pri pretoku 0,8 ml/min. Komponente v ekstraktu smo identificirali s primerjavo UV spektrov in retenzijskih časov referenčnih substanc, vsebnost komponent pa smo določili na podlagi umeritvene krivulje.
Pokazali smo linearnost metode za določevanje klorogene kisline, eriocitrina in hesperidina v območju 2,5–300 g/ml, kavne kisline v območju 1,0–300 g/ml in rutina v koncentracijskem območju 5,0–300 g/ml. Določili smo mejo zaznave (LOD), ki za vse komponente znaša 1,0 g/ml ter mejo določljivosti (LOQ), ki je 2,5 g/ml za hesperidin, eriocitrin in klorogeno kislino, 1,0 g/ml za kavno kislino ter 5,0 g/ml za rutin. Rezultati kažejo, da je metoda natančna za določevanje vseh komponent, medtem ko je točna za določevanje hesperidina in kavne kisline.
Z optimizirano metodo smo določili vsebnost posameznih antimikrobnih komponent v različnih vzorcih ekstrakta limone. Vse komponente smo zaznali v liofiliziranem suhem ekstraktu limone, v katerem sta prevladovala hesperidin (7,09 mg/g ekstrakta) in eriocitrin (3,81 mg/g ekstrakta). Visoke vsebnosti hesperidina smo zaznali tudi v liofiliziranem ekstraktu limete, kjer je ta vrednost znašala 9,74 mg/g ekstrakta. Ostale komponente so bile v teh in ostalih vzorcih zaznane v manjših količinah, nekje od 0,15 do 2,5 mg komponente/g ekstrakta.Hesperidin, eriocitrin, rutin, caffeic acid and chlorogenic acid are antimicrobial substances found in lemon and its dry extracts. Due to their positive effects, especially on human health, they are increasingly used in various dietary supplements. Development and optimization of the HPLC method was needed in order to identify and quantify these antimicrobial substances in dried lemon extracts.
In order to separate the compounds, gradient elution was used, in which an increasing amount of organic phase (methanol) was gradually added to 0.4% acetic acid at a flow rate of 0.8 ml/min. The components in the extract were identified by comparing the retention times, and the component content was determined based on the calibration curve.
We showed the linearity of the method for the determination of chlorogenic acid, eriocitrin and hesperidin in the range from 2.5 to 300 μg/ml, caffeic acid in the range from 1 to 300 μg/ml and rutin in the concentration range from 5 to 300 mg/ml. The limit of detection (LOD) value was 1m μg/ml for all components and the limit of quantification (LOQ) values were 2.5 μg /ml for hesperidin, eriocitrin and chlorogenic acid, 1 μg /ml for caffeic acid and 5 μg /ml for rutin. The results showed that the method is precise for the determination of all components, while it is accurate for the determination of hesperidin and caffeic acid.
The final method was used to determine the content of individual antimicrobial components in the samples. All components were detected in lyophilized dried lemon extract, in which hesperidin (7.09 mg/g extract) and eriocitrin (3.81 mg/g extract) predominated. High levels of hesperidin were also detected in the lyophilized lime extract, where this value was 9.74 mg/g of extract. Other components were also detected in samples, in which quantities were somewhere between 0.15 and 2.5 mg of component/g of extract
