Korelacija strukture i hromatografskog ponašanja polioksigenovanih steroida u uslovima planarne hromatografije

Abstract

U okviru ove doktorske disertacije detaljno je proučavan uticaj supstituenata vezanih za A prsten na hromatografsko ponašanje serije polioksigenovanih steroida u uslovima tankoslojne hromatografije. U cilju proučavanja uticaja polarnosti sorbenta na retenciju ispitivanih jedinjenja upotrebljena su dva veoma različita sorbenta: polarni nemodifikovani silika-gel i nepolarni C-18 modifikovani silika-gel. Posebna pažnja posvećena je razmatranju uticaja sastava primenjenih mobilnih faza na retenciju radi što boljeg razumevanja odnosa hromatografskog ponašanja i lipofilnosti ispitivanih jedinjenja. Uzimajući u obzir svojstva sorbenta kao i korišćenih mobilnih faza, retenciono ponašanje je izučavano u uslovima normalno-fazne i reverzno-fazne hromatografije. Kao tipični normalnofazni sistemi upotrebljeni su polarni silika-gel kao stacionarna faza i manje polarni rastvarači (aceton, heksan, acetonitril, dihlormetan) kao mobilna faza. Reverzno-fazni sistemi sastojali su se od smeše voda ˗ organski rastvarač (aceton, metanol ili acetonitril) i oktadecil modifikovanog silika-gela kao stacionarne faze. U uslovima normalno-fazne hromatografije sistem aceton/nheksan se pokazao kao pogodan za ispitivanje uticaja sastava mobilne faze na hromatografsko ponašanje polioksigenovanih steroidnih jedinjenja. U uslovima reverzno-fazne hromatografije najbolja selektivnost je postignuta primenom mobilne faze metanol - voda. U okviru ovog rada proučena je i mogućnost primene tankoslojne hromatografije za brzo i jednostavno određivanje lipofilnosti kao važnog parametra biološke aktivnosti ispitivanih steroida. Utvrđeno je da postoji linearna zavisnost između retencionih parametara ispitivanih jedinjenja i sastava dvokomponetnih mobilnih faza. Ispitivanjem korelacije između hromatografski određenih parametara lipofilnosti i teorijski izračunatih logP vrednosti utvrđeno je da je reverzno-fazna hromatografija pogodnija za procenu lipofilnosti ispitivanih jedinjenja.Takođe pokazano je da ispitivani polioksigenovani steroidi sa supstituisanim A prstenom predstavljaju kongenernu seriju. Primenom višestruke linearne regresije (multiple linear regression, MLR) i metode parcijalne regresije najmanjih kvadrata (partial least squares, PLS) proučavane su moguće zavisnosti između hromatografskog ponašanja i molekulskih svojstava analita. Na osnovu statističkih parametara dobijenih za tri reverzno-fazna hromatografska sistema najbolje korelacije između RM 0 i deskriptora bile su u sistemu C-18 / metanol-voda što izdvaja ovaj hromatografski sistem kao najpogodniji za određivanje lipofilnosti analita. Na osnovu regresionih modela određeni su deskriptori koji najbolje opisuju ponašanje analita u odnosu na utvrđene retencione parametre. Dobijeni su statistički značajni modeli kvantitativnog odnosa strukture i retencije (QSRR). Izračunata lipofilnost izražena kao XlogP, površinski napon (ST) i Hansenovo vodonično vezivanje (HHB) su deskriptori koji najbolje opisuju QSRR i u višestrukoj linearnoj regresiji i u parcijalnoj regresiji najmanjih kvadrata.

The framework of this thesis was to thoroughly investigate influence of structural moiety attached to A ring on chromatographic behavior of series polyoxigenated steroids under thin-layer chromatography conditions. In order to examine the effect of sorbent polarity on retention behavior of the investigated substances, two different sorbents were selected: polar unmodified silica gel and non-polar C-18 modified silica. In addition, influence of the mobile phase composition was discussed with view to determine relationship between chromatographic behavior and lipophilicity. Taking into account the nature of both sorbents and chromatographic solvents used, the chromatographic behavior of the substances was investigated under normal- and reversed-phase conditions. Polar silica gel with less polar solvents was used as typical normal-phase systems, whereas alkyl modified silica gel (RP-18 silica) in combination with polar water–organic mobile phases (methanol, acetone or acetonitrile) - for typical reversed-phase separation. It was found that in normal-phase chromatography acetone/n-hexane was more suitable for studying relationship between chromatographic behavior of the polioxigenated steroids and mobile phase composition. Under reversed-phase condition the best selectivity was achieved by the use of methanol-water mobile phase. An additional goal of this thesis was to investigate possibility of application of thin-layer chromatography for rapid and simple determination of lipophilicity as parameter of special importance for biologically active substances. A linear relationship between retention parameters of the investigated compounds and composition of the binary mobile phase used was established. On the basis of correlation between chromatographically determined lipophilicity and calculated logP values it was found that reversed-phase chromatography is useful method for lipophilicity estimation. In addition, it is appear that investigated polyoxigenated steroids represent a congeneric series of compounds. Chromatographically determined parameters of lipophilicity (RM 0) were used for developing appropriate models for structure-retention relationships by multivariate statistical analysis. The retention data for system methanol-water on C-18 silica phase has the most significant correlation between RM 0 and descriptors indicating this chromatographic system as most suitable for lipophilicity determination of the studied compounds. Retention data were correlated to molecular characteristics of the analytes with view to examine possible relationships by the means of multiple linear regression and partial least square regression. On the basis of comparison of the statistical parameters obtained for both multiple linear regression and partial least square regression models, descriptors best describing the analyte behavior were selected. Statistically significant and physically meaningful structure-retention relationships were obtained. Calculated lipophilicity expressed as XlogP as well surface tension and Hansen hydrogen bonding was included in both multiple linear regression and partial least square regression models.