Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography.

Three polysaccharide-based chiral stationary phases, Sepapak(®) 1, Sepapak(®) 2 and Sepapak(®) 3 have been evaluated in the present work for the stereoisomer separation of a group of 12 flavonoids including flavanones (flavanone, 4'-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, hesperetin, naringenin) and flavanone glycosides (hesperidin, naringin) by nano-liquid chromatography (nano-LC). The behaviour of these chiral stationary phases (CSPs) towards the selected compounds was studied in capillary columns (100µm internal diameter (i.d.)) packed with the above mentioned CSPs using polar organic, reversed and normal elution modes. The influence of nature and composition of the mobile phase in terms of concentration and type of organic modifier, buffer type and water content (reversed phase elution mode) on the enantioresolution (R(s)), retention factor (k) and enantioselectivity (α) was evaluated. Sepapak(®) 3 showed the best chromatographic results in terms of enantioresolution, enantioselectivity and short analysis time, employing a polar organic phase mode. A mixture of methanol/isopropanol (20/80, v/v) as mobile phase enabled the chiral separation of eight flavanones with enantioresolution factor (R(s)) in the range 1.15-4.18. The same analytes were also resolved employing reversed and normal phase modes with mixtures of methanol/water and hexane/ethanol at different ratios as mobile phases, respectively. Loss in resolution for some compounds, broaden peaks and longer analysis times were observed with these last two chromatographic elution modes. Afterwards, a comparison with the other two CSPs was performed. A lower discrimination ability of Sepapak(®) 1 and Sepapak(®) 2 towards all the studied flavanoids was observed. However, Sepapak(®) 1 allowed the separation of naringenin enantiomers and naringin stereoisomers in polar organic phase which were not resolved with the other two CSPs. The nature of the chiral selector was found to be of utmost importance for the resolution of the selected compounds. Indeed, significant differences in enantioresolution among the three tested CSPs were observed. With regard to the only few data reported in the literature for the resolution of this class of compounds using polysaccharide-based CSPs by high performance liquid chromatography (HPLC), the results obtained in this study by means of nano-LC showed higher (R(s)) values and shorter analysis time.

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode.

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 µm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50°C. Linear correlation of lnk vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation. To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-beta-cyclodextrin chiral stationary phase.

In this paper a phenyl-carbamate-propyl-beta-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4'-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R(s)), retention factor (k) and enantioselectivity (alpha) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2'-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H(2)O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H(2)O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2'-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.

Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography.

Hesperetin (HT) is a flavanone abundantly found in citrus fruits. It has been reported that HT possesses significant antioxidant, anticancer, anti-inflammatory and analgesic activities. This explains the necessity of developing new methods more powerful and sensitive for analyzing HT in biological fluids. Taking into account the chiral nature of HT, the study of the stereospecific kinetics of in vitro and in vivo metabolism and tissue distribution could be a useful tool for further understanding stereoselective biotransformations in human body. A simple nano-liquid chromatographic method for the determination of the enantiomeric composition of hesperetin in human urine was developed. Chiral separation was achieved using a 100 microm I.D. capillary, packed with phenyl-carbamate-propyl-beta-cyclodextrin stationary phase, employing a mobile phase composed by a mixture of triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) at room temperature. The detection was done by using on-column UV detector at 205 nm. Calibration curves were linear in the studied concentration range from 0.25 to 25 microg/mL (r(2)>0.999). Precision assay was <4.5% and was within 3% at the limit of quantification (0.5 microg/mL). The recovery of 7-ethoxycoumarin (IS), R- and S-hesperetin was greater than 82.48%, utilizing a liquid-liquid extraction procedure. The developed method was successfully applied to the determination of hesperetin enantiomers in urine samples obtained from a male volunteer, after the ingestion of 1L of a commercial blood orange juice.

Preparation and enantioseparation characteristics of three chiral stationary phases based on modified beta-cyclodextrin for liquid chromatography.

In order to study the effect of the nature and the length of the spacer, three mixed 10-undecenoate/phenylcarbamate derivatives of beta-cyclodextrin have been prepared and linked to allylsilica gel by means of a radical reaction. The chiral recognition ability of the resulting materials, when used as liquid chromatography chiral stationary phases (CSPs), was evaluated using heptane and either 2-propanol or chloroform as organic mobile-phase modifiers. A large variety of racemic compounds have been separated successfully on these CSPs (mainly pharmaceuticals and herbicides). Optimization of these separations was discussed in terms of mobile-phase composition and structural patterns of the injected analytes. The efficiencies of the three prepared materials were compared to those of previously described perphenylated-beta-cyclodextrin column and to analogous cellulose derivative-based CSPs.

Preparation and chromatographic properties of a multimodal chiral stationary phase based on phenyl-carbamate-propyl-beta-CD for HPLC.

A phenylcarbamate derivative of 2-hydroxypropyl-beta-CD bonded stationary phase was prepared by a previously described method. Its enantiomeric recognition abilities were evaluated as chiral stationary phase (CSP) in normal, polar organic and RP conditions by HPLC. The relevant structural features of the prepared stationary phase which make it an effective chiral selector are discussed. This material seems to have an excellent enantioselectivity for a variety of racemic analytes in the three modes. Hence it can be considered a highly effective multimodal column. Retention factor (k), selectivity (alpha) and resolution (R(s)) were the chosen parameters to describe the column performance. Optimization of these separations was discussed in terms of mobile phase composition, flow rate and structural patterns of the injected analytes.