The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.
- MeSH
- acetonitrily chemie MeSH
- aza sloučeniny * analýza chemie MeSH
- fluorescenční barviva * analýza chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- molekulární sondy * analýza chemie MeSH
- oligodeoxyribonukleotidy * analýza chemie MeSH
- rozpouštědla MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Creatine, phosphocreatine, and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on RP silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography (HILIC). This study describes a first systematic investigation of the ability of ZrO2 to separate creatine, phosphocreatine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate and compares the results with those obtained on TiO2 . All analytes showed a HILIC-like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in organic-rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO2 as a promising phase for HILIC of biological samples that deserves further investigation.
- MeSH
- adenosindifosfát chemie izolace a purifikace metabolismus MeSH
- adenosinmonofosfát chemie izolace a purifikace metabolismus MeSH
- adenosintrifosfát chemie izolace a purifikace metabolismus MeSH
- biologické markery chemie metabolismus MeSH
- chromatografie kapalinová MeSH
- fosfokreatin MeSH
- hydrofobní a hydrofilní interakce MeSH
- kreatin chemie izolace a purifikace metabolismus MeSH
- myokard chemie metabolismus MeSH
- zirkonium chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Benfluron (B) [5-(2-dimethylaminoethoxy)-7H-benzo[c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway (O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm x 4.6 mm ID, 5 microm) using a mobile phase acetonitrile-1 M NaClO4 (40:60, v/v). Another mobile phase, methanol-1 M NaClO4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min(-1) in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.
- MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- chromatografie kapalinová metody MeSH
- druhová specificita MeSH
- financování organizované MeSH
- fluoreny analýza metabolismus MeSH
- hospodářská zvířata MeSH
- játra chemie metabolismus MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- lidé středního věku MeSH
- lidé MeSH
- molekulární konformace MeSH
- morčata MeSH
- potkani Wistar MeSH
- preklinické hodnocení léčiv metody MeSH
- protinádorové látky analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- lidé středního věku MeSH
- lidé MeSH
- morčata MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
