Razvoj metode tekočinske kromatografije za ugotavljanje vsebnosti orientina, izoorientina in viteksina v rastlinskih vzorcih

Bogataj, Anamarija (avtor), Tavčar, Eva (mentor), Kokalj Ladan, Meta (komentor)

Flavonoidi so sekundarni rastlinski metaboliti, ki se v kozmetičnih izdelkih uporabljajo kot kozmetično aktivne sestavine zaradi njihovega antioksidativnega in protivnetnega delovanja. Antioksidanti so v kozmetičnih izdelkih pomembni tudi za podporo fizikalno kemijske stabilnosti končnega izdelka. Namen diplomskega dela je bil razviti ustrezno metodo tekočinske kromatografije visoke ločljivosti, s katero bi lahko v različnih rastlinskih vzorcih analizirali in kvantificirali flavonoide: orientin, izoorientin in viteksin. S spreminjanjem časa trajanja metode in deleža naraščanje mobilne faze smo razvili metodo, s katero lahko kvantificiramo izbrane flavonoide. Metodo smo delno validirali in določili linearnost metode, točnost, natančnost, selektivnost, ponovljivost, določili pa smo tudi mejo detekcije in kvantifikacije. Mobilna faza je sestavljena iz različnih deležev zmesi A in B. Zmes A smo pripravili iz vode, acetonitrila in trifluorocetne kisline (TFA) v razmerju 1000:20:1. V odstotkih navajamo delež zmesi B, ki je sestavljena iz acetonitrila, vode in TFA v razmerju 1000:20:1 po gradientnem programu s pretokom 2 mL/min: 0,01-1 min 0 %, 0-1,01 min 3 %, 1,01-45 min 8 %, 45-55 min 38 %, 55-55,01 min 100 %, 55,01-58 min 100 %, 58,01-60 min 0 %. Vzorce smo injicirali v reverzno fazno kolono v količini po 2 µL in jih detektirali pri valovni dolžini 254 nm. Selektivnost metode smo potrdili s primerno ločbo analitov na kromatogramu in ujemanju njihovih retencijskih časov s standardnimi referenčnimi spojinami. Linearnost smo potrdili s korelacijskim koeficientom, ki je pri rutinu, orientinu, izoorientinu in viteksinu znašal R2 ≥ 0,9993. Povprečne vrednosti točnosti so bile znotraj intervala ± 10 % po postopku priprave s steklenimi čolnički za kontrolne vzorce rutina nižjih koncentracij (94,903 %) ter po postopku priprave s plastičnimi čolnički, za kontrolne vzorce rutina nižjih koncentracij (92,64 %) in kvercetina (95,953 %). Ponovljivost ni odstopala od zgornje meje 5 % po postopku priprave s steklenimi čolnički za kontrolne vzorce rutina nižjih koncentracij (RSD = ± 1,38 %) ter po postopku priprave s plastičnimi čolnički, za kontrolne vzorce rutina nižjih koncentracij in kvercetina (RSD = ± 2,992 %, RSD = ± 3,531 %).

• Jezik: Slovenski jezik
• Ključne besede: flavonoidi, validacija, vsebnost, tekočinska kromatografija visoke ločljivosti
• Vrsta gradiva: Diplomsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-173400
• Datum objave v RUL: 17.09.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of a liquid chromatography method for determining the contents of orientin, isoorientin and vitexin in plant samples

Izvleček

Flavonoids are secondary plant metabolites used in cosmetic products as cosmetically active ingredients due to their antioxidant and anti-inflammatory properties. Antioxidants are also important in cosmetic products for supporting the physical and chemical stability of the final product. The aim of this thesis was to develop a suitable high-performance liquid chromatography method for the detection and quantification of the flavonoids orientin, isoorientin and vitexin in various plant samples. By changing the duration of the method and the rate of increase of the mobile phase, we developed a method for quantifying the selected flavonoids. We partially validated the method and determined its linearity, accuracy, precision, selectivity and repeatability, and we also determined the detection and quantification limits. The mobile phase consisted of different proportions of mixtures A and B. Mixture A was prepared from water, acetonitrile, and trifluoroacetic acid (TFA) in a ratio of 1000:20:1. The percentage of mixture B, which consists of acetonitrile, water, and TFA in a ratio of 1000:20:1, is given in a gradient program with a flow rate of 2 mL/min: 0,01-1 min 0 %, 1-1,01 min 3 %, 1,01-45 min 8 %, 45-55 min 38 %, 55-55,01 min 100 %, 55,01-58 min 100 %, 58,01-60 min 0 %. The samples were injected into a reverse phase column in quantities of 2 µL and detected at a wavelength of 254 nm. Selectivity was confirmed by adequate separation of analytes on the chromatogram and matching of their retention times with standard reference compounds. The linearity of the method was confirmed by a correlation coefficient R2 ≥ 0.9993 for rutin, orientin, isoorientin and vitexin. The average accuracy values were within the range ± 10 % after preparation with glass boats for control samples of lower concentrations (94.903 %) and after preparation with plastic boats for control samples of lower concentrations (92.64 %) and quercetin (95.953 %). Repeatability did not deviate from the upper limit of 5 % after the preparation procedure with glass boats for control samples of lower concentrations of rutin (RSD = ± 1.38 %) and after the preparation procedure with plastic boats, for control samples of lower concentrations of rutin and quercetin (RSD = ± 2.992 %, RSD = ± 3.531 %).

• Ključne besede: flavonoids, validation, content, high-performance liquid chromatography