A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.

• MeSH
• cholestadienoly chemie   izolace a purifikace   MeSH
• cholesterol analogy a deriváty   chemie   izolace a purifikace   MeSH
• chromatografie s reverzní fází metody   normy   MeSH
• fytosteroly chemie   izolace a purifikace   MeSH
• ketocholesteroly chemie   izolace a purifikace   MeSH
• lněný olej analýza   chemie   MeSH
• oleje rostlin analýza   chemie   MeSH
• referenční standardy MeSH
• sitosteroly chemie   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   normy   MeSH
• stigmasterol chemie   izolace a purifikace   MeSH
• stříbro chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Retention characteristics of selected synthetic 5'-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.

• MeSH
• chromatografie kapalinová metody   MeSH
• chromatografie s reverzní fází metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• oligonukleotidy chemická syntéza   chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH