Carlo Bertucci

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

SummaryA new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.

Cinchona alkaloids for preparing new, easily accessible chiral stationary phases.I. 11-(10,11-Dihydro-6′-methoxy-cinchonan-9-OL)-tiopropylsilanized silica.

Abstract By reaction of mercatopropylsilanized silica with quinine in the presence of AIBN as radical initiator, a silica supported alkaloid can be obtained, which is effective in the HPLC resolution of racemic arylalkylcarbinols and binaphtol derivatives.

Cinchona alkaloids for preparing new, easily accessible chiral stationary phases : II. Resolution of binaphthol derivatives on silica-supported quinine☆

Abstract The preparation of the new chiral stationary phase SiSQuin, obtained by reaction of γ-mercaptopropylsilanized silica with the commercially available Cinchona alkaloid quinine, is described. This support is efficient for the rapid (6–7 min) separation of some binaphthol derivatives (separation factors between 1.08 and 1.16). The use of a circular dichroism detector allows the elution order to be established in a non-empirical way. This information is then used to propose a chiral recognition mechanism.

The binding of 5-fluorouracil to native and modified human serum albumin : UV, CD, and 1H and 19F NMR investigation

5-Fluorouracil (FU) is an important and widely used antineoplastic drug that is carried in the serum by plasma proteins. Protein binding studies of this drug to human serum albumin (HSA) have been carried out by several spectroscopic techniques. Difference circular dichroism and UV studies provided information on the class of binding sites involved in the interaction. In particular, displacement experiments showed that FU has at least one secondary binding site in the coumarin binding area, but does not interact with the benzodiazepine binding area. Binding was also investigated by difference 1H NMR and by measuring the increase in the 19F NMR signal of FU when bound to HSA. Finally, evidence was obtained that chemical acetylation of Lys199 results in a decreased apparent binding affinity constant (nK) for FU. Such a modification is induced under physiological conditions by aspirin.

Analysis of ACE-inhibitors by CE using alkylsulfonic additives.

Capillary electrophoresis (CE) was applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in pharmaceuticals (tablets). Since a free solution CE system failed to reach a complete separation of closely related compounds (lisinopril, ramipril, benazepril, quinapril), alkylsulfonic additives (sodium heptansulfonate and (+)-10-camphorsulphonic acid) were added to the running buffer: improved separations were obtained suggesting a favourable effect of ion-pairing interactions between analytes and additives. The separations were carried out in acidic medium and a systematic investigation of electrophoretic parameters was made to evaluate the performance of the selected additives. Under the optimized conditions, ramipril and benazepril in their commercial dosage forms were determined confirming the applicability of the developed CE approach to the analysis of pharmaceutical samples; the results were also compared with those obtained applying a previously described and validated HPLC method.

Chiral stationary phases based on cinchona alkaloids for the HPLC resolution of racemates

SummaryThe employment of three chiral stationary phases (CSPs) obtained by derivatizing γ-mercaptopropyl-silanized silica gel with quinine, quinidine and N-methyl-quinium iodide, for the separation of organic racemates, is presented. They are quite useful in the resolution of alkylarylcarbinols, binaphthyl derivatives, amides and other substances of pharmaceutical interest.

Ligand binding to a human serum albumin stationary phase: use of same-drug competition to discriminate pharmacologically relevant interactions.

A technique based on a human serum albumin (HSA) stationary phase high-pressure liquid chromatography (HPLC) has been successfully used for the past few years to characterize the interactions between HSA and new substrates. Immobilized HSA conserves the binding properties of the protein in solution, allowing fast and reliable analyses of binding interactions. Nevertheless, clear evidence that all binding mechanisms of HSA-HPLC are pharmacologically relevant is so far lacking. In particular, non-stoichiometric interactions of injected ligands with stationary phase components such as silica and the amino acid medium (other than protein binding areas) might interfere with the correlation between chromatographic retention and HSA binding. Here we present a quantitative method to distinguish between the molecular interactions of a ligand with binding areas of potential pharmacological interest and other, non-saturable binding mechanisms. Such a method, based on HPLC same-ligand displacement, is simple and reliable, as confirmed by in situ protein denaturation. Consequently, we were able to distinguish between different types of competitions detected in the co-binding of two drugs to HSA.

Site I on human albumin: Differences in the binding of (R)- and (S)-warfarin

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.

Improved chromatographic performance of a modified human albumin based stationary phase.

Derivatization of the free cys34 in human serum albumin (HSA) anchored to a silica matrix has been performed by in situ reaction with ethacrynic acid. This modification, which is reported to occur under physiological conditions, gives rise in practice to a new column with different binding properties with respect to the column based on the native protein. Significant differences were observed in the binding of drugs known to bind to site I, (R)-(S)-warfarin and phenylbutazone, and to site II, 1,4-benzodiazepin-2-ones and nonsteroidal anti-inflammatory agents. In particular, the chromatographic retentions markedly decreased for most of the drugs, and, in the case of chiral compounds, significant differences were often observed in the behavior of the two enantiomers, with higher values of enantioselectivity obtained for some of the examined compounds. Furthermore, the noncovalent binding of ethacrynic acid to the protein modifies the binding properties of the albumin.

Determination of optical purity by high-performance liquid chromatography upon non-chiral stationary phases with dual circular dichroism/absorption detection

The application of a circular dichroism (CD)-based detection system in HPLC using a non-chiral stationary phase is presented. The simultaneous measurement of CD, absorbance and anisotropy factor allows the determination of optical purity and the assignment of the absolute configuration to the prevailing enantiomer. This represents a further application of CD to analyse an optically active compound. The reliability of the method and the detection limit depend on the value of the anisotropy factor and on the CD intensity, respectively.