An integrated on-chip sirtuin assay.

A microchip-based assay to monitor the conversion of peptide substrates by human recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused silica microchip consisting of a microfluidic separation structure with an integrated serpentine micromixer has been used. As substrate for the assay, we used a 9-fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known substrate of hSIRT1. The Fmoc group at the N-terminus resulting from solid-phase peptide synthesis enabled deep UV laser-induced fluorescence detection with excitation at 266 nm. The enzymatic reaction of 0.1 U/µL hSIRT1 was carried out within the serpentine micromixer using a 400 µM solution of the peptide in buffer. In order to reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose. The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The approach was successfully utilized to screen various SIRT inhibitors.

Simultaneous determination of dextromepromazine and related substances 2-methoxyphenothiazine and levomepromazine sulfoxide in levomepromazine on a cellulose tris(4-methylbenzoate) chiral column.

A high-performance liquid chromatography method for the determination of dextromepromazine, levomepromazine sulfoxide and 2-methoxyphenothiazine in levomepromazine samples was developed. The separation of the analytes was achieved within 10 min on a stationary phase containing cellulose tris(4-methylbenzoate) as chiral selector. The mobile phase consisted of 0.1% diethylamine in methanol with a flow rate of 1.0 mL/min. The method was validated according to the International Council for Harmonization guideline Q2(R1). The detection limits based on a signal-to-noise ratio of 3 were in the range of 0.002 to 0.005 µg/mL. The method proved to be precise and accurate in the concentration range of 0.025-1.0 % for levomepromazine sulfoxide and 2-methoxyphenothiazine and 0.025% to 3.0% for dextromepromazine relative to a concentration of 0.1 mg/mL of levomepromazine, with the exception of levomepromazine sulfoxide at the 0.1% level. The method was subsequently applied to the analysis of finished pharmaceutical products as well as of reference substances of the European Pharmacopoeia.

Chiral recognition in separation science: an overview.

Chiral recognition phenomena play an important role in nature as well as analytical separation sciences. In separation sciences such as chromatography and capillary electrophoresis, enantiospecific interactions between the enantiomers of an analyte and the chiral selector are required in order to observe enantioseparations. Due to the large structural variety of chiral selectors applied, different mechanisms and structural features contribute to the chiral recognition process. This chapter briefly illustrates the current models of the enantiospecific recognition on the structural basics of various chiral selectors.

Linear free energy relationship analysis of retention factors in cerasome electrokinetic chromatography intended for predicting drug skin permeation.

The retention factors of neutral, positively charged, and negatively charged solutes were determined in a liposome electrokinetic chromatography (EKC) system, where cerasome was used as the investigated liposome. The Abraham linear free energy relationship (LFER) for neutral and ionized solutes gave a good account of the retention factors (N = 71, R(2) = 0.814, and SD = 0.29 log units). It was shown that the calculated retention factors for 16 neutral acids were about four times higher than those of the corresponding anions, whereas the calculated retention factors for neutral bases were less than those for the corresponding cations by a factor of 0.36. The LFER equation for neutral species, anions, and cations was compared with those for partition from water into a number of solvents and for n-octanol-water distribution coefficients. It was shown that the cerasome EKC system is substantially different to the other systems and consequently it could be a very useful additional model system, possibly for predicting skin permeation. It was further shown that there are considerable advantages in the use of Abraham LFERs that can encompass not only neutral molecules but also ionic species.