Karin Balmér

Reversed retention order and other stereoselective effects in the separation of amino alcohols on Chiralcel OD

Abstract The enantioselective resolution of a series of amino alcohols on Chiralcel OD was studied with respect to the effect of temperature, alcohol additive and water content in a mobile phase of hexane with added diethylamine. The chain length between the hydroxy and the amino groups in the solutes had a considerable influence on the stereoselectivity. For one of the amino alcohols a reversal of the retention order between the antipodes was obtained by varying the above parameters. The amino alcohols are retained by at least two chiral sites, one of which is highly dependent on hydrogen bonding. This bonding ability can be directly controlled by the water content of the mobile phase.

Stereoselective effects in the separation of enantiomers of omeprazole and other substituted benzimidazoles on different chiral stationary phases

Abstract The enantioselective separation of omeprazole on different chiral stationary phases was investigated. The two enantiomers could be resolved on three different phases with immobilized protein, Chiral-AGP, Ultron ES-OVM and BSA-DSC, employing aqueous mobile phases with 2-propanol as organic modifier. On Chiralpak AD, an amylose-based chiral stationary phase, the enantiomers of omeprazole and three analogues could be separated using a non-polar hexane-ethanol mobile phase. For omeprazole the retention order was reversed when 2-propanol was replaced with ethanol or methanol as the modifier of hexane in the mobile phase.

Liquid chromatographic separation of the enantiomers of metoprolol and its α-hydroxy metabolite on Chiralcel OD for determination in plasma and urine

Abstract The two enantiomers of metoprolol and the four enantiomeric forms of α-hydroxymetoprolol were separated by liquid chromatography on a Chiralcel OD column containing a cellulose tris(3,5-dimethyl-phenylcarbamate) chiral stationary phase. The column efficiency was strongly dependent on the flow-rate and the enantioselectivity was influenced by temperature. Of utmost importance for the chiral separation was the water content of the mobile organic phase. The separation system was used for the separation and determination of the enantiomers in plasma and urine samples. The metoprolol enantiomers could be determined by fluorescence down to 10 nmol/1 of each in plasma with a relative standard deviation of less than 15%.

The neuropeptide Y (NPY) Y1 receptor antagonist BIBP 3226: effects on vascular responses to exogenous and endogenous NPY in the pig in vivo

The antagonistic effects of the neuropeptide Y (NPY) Y1 receptor antagonist BIBP 3226 on equally prominent vascular responses evoked by sympathetic nerve stimulation and exogenous NPY were compared during different intravenous (i.v.) infusions of the antagonist (0.19–190 nmol kg−1 min−1, for 30 min). High frequency sympathetic nerve stimulation in reserpine‐treated pigs in vivo evoked non‐adrenergic vasoconstrictor responses in kidney and hind limb, in the latter followed by a long‐lasting phase of decreased blood flow. The vascular response in the kidney and the long‐lasting phase in hind limb resembled the effects of exogenous NPY administered i.v. (kidney) and intraarterially (i.a.) (in the hind limb, which only responded to higher NPY doses). Plasma levels of BIBP 3226 reached a plateau within 20 min and the inhibitory effects on vascular responses were studied during the last ten minutes of infusion. The elimination of BIBP 3226 from plasma was found to fit a two‐compartment model with an α‐phase of 2.0±0.2 min and a β‐phase of 20.1±0.9 min. Significant inhibition of presumably Y1 receptor‐mediated vascular responses evoked by both endogenous and exogenous NPY in kidney and hind limb was seen even during low‐dose infusion of BIBP 3226 (1.9 nmol kg−1 min−1), when plasma levels of the antagonist reached 59±8 nM. The maximum inhibitory effects of BIBP 3226 were seen during the highest‐dose infusion (190 nmol kg−1 min−1), when the long‐lasting vasoconstrictor responses in hind limb to sympathetic nerve stimulation and exogenous NPY administration (i.a.) were abolished. Simultaneously, the vascular responses in kidney to exogenous NPY were inhibited by 89% and to sympathetic nerve stimulation by 60%. It is concluded that BIBP 3226 has a short half‐life in plasma and should preferably be given by i.v. infusions to maintain blockade and avoid non‐specific effects. Furthermore, BIBP 3226 dose‐dependently and with similar potency antagonizes vascular responses to exogenous and endogenous NPY both in the kidney and hind limb of the reserpine‐treated pig in vivo. Thus, inhibition of vascular responses to exogenous NPY may be a good indicator of the dose of this antagonist needed to inhibit sympathetic Y1 receptor‐transmission.

Optimization of detection sensitivity for enantiomers of metoprolol on silica-bonded α1-acid glycoprotein

Abstract On the chiral phases CHIRAL-AGP and EnantioPac, organic modifiers, pH and temperature have been varied with isocratic and gradient techniques to obtain minimum retention and maximum peak height by baseline resolution of R-and S-metoprolol. The decrease in retention obtained by gradient elution gives, by limiting baseline resolution, 20–60% greater peak heights than those obtained by the isocratic technique. A reduction of the retention by a change in the pH is more favourable than addition of modifiers such as acetonitrile or 2-propanol. It gives a smaller decrease in resolution by isocratic elution and a pH gradient can give considerably stronger peak compression than an acetonitrile gradient as indicated by an estimation of the detection limits. For metoprolol enantiomers, CHIRAL-AGP gives about three times lower reduced plate heights than does EnantioPac.

Fully automated gradient elution liquid chromatographic assay of omeprazole and two metabolites

An automated liquid chromatographic method for the determination of omeprazole and two metabolites in plasma and urine is described. It utilizes the Technicon Fully-Automated-Sample-Treatment-LC system (FAST(R)-LC). Sample preparation is achieved by air-segmented continuous-flow providing solvent extraction, evaporation to dryness and reconstitution before injection onto a reversed-phase column. The compounds are separated by isocratic or gradient elution with acetonitrile-phosphate buffer mobile phases and quantified by UV-measurements at 302 nm. The limit of determination (relative standard deviation 10-15%) is 50 nmol l(-1) in plasma (800 microl) and 200 nmol l(-1) in urine (200 microl). The sample capacity is six or three samples per hour, depending on the elution mode.

In vivo characterization of the novel neuropeptide Y Y1 receptor antagonist H 409/22.

We studied the effects of the novel neuropeptide Y (NPY) Y1 receptor antagonist H 409/22, and its inactive enantiomer H 510/45, on vascular responses evoked by endogenous and exogenous NPY in the pig in vivo. H 409/22 and H 510/45 were given as 30-min infusions, and the antagonistic effects and circulating plasma concentrations were measured. The initial and terminal half-lives of H 409/22 in plasma were approximately 3 and 30 min, respectively. In pigs pretreated with reserpine and transection of sympathetic nerves (depletion of noradrenaline), sympathetic nerve stimulation evoked nonadrenergic vasoconstrictor responses in kidney and hindlimb, mediated by neuronally released NPY. Significant inhibition of these vasoconstrictor responses, as well as of vascular responses to injections of exogenous NPY, were seen during a low-dose infusion of H 409/22 (1.8 nmol/kg/min), when plasma levels of the antagonist reached 77 +/- 8 nM. Greatest inhibitory effects were seen at the highest dose of H 409/22 (180 nmol/kg/min, giving plasma levels of 7.4 +/- 0.6 microM) when all vascular responses evoked by NPY were strongly attenuated or largely abolished. H 510/45 did not affect any of the vascular responses studied. It is concluded that H 409/22 potently and dose-dependently antagonizes vascular responses to exogenous and endogenous NPY in the pig, and thus represents an interesting tool for studies on NPY Y1 receptor-mediated effects in vivo.

Determination of metaprolol and two major metabolites in plasma and urine by column liquid chromatography and fluorometric detection

Metoprolol and its alpha-hydroxy metabolite were determined in plasma down to 2 nmol/l (S.D. 10-15%) after solvent extraction and bonded-phase liquid chromatography with fluorometric detection. The major metabolite with a carboxylic function was also measured in plasma when liquid-solid extraction on a column activated with dodecyl sulphate was applied. In urine the three components were assayed by direct injection of a diluted sample.

Direct chiral separation of almokalant on Chiralcel OD and Chiralpak AD for liquid chromatographic assay of biological samples

The four isomers of almokalant, a new antiarrhythmic substance under investigation, were separated by liquid chromatography on a Chiralcel OD and a Chiralpak AD column containing cellulose and amylose tris(3,5-dimethylphenylcarbamate), respectively. Both chiral stationary phases separate almokalant into the four isomers, but the retention orders are different if the carbamate is derivatized on cellulose or amylose. The Chiralcel OD column was used for the separation and determination of the isomers in urine at levels down to 100 nmol/l for the first three eluted and 200 nmol/l for the last with a relative standard deviation of less than 15%. The fluorescence response was increased by post-column ionization after stereoselective separation on the Chiralpak AD column. The isomers of almokalant could be determined at levels down to 10 nmol/l in plasma with a relative standard deviation of less than 15%.

CHROMATOGRAPHIC RESOLUTION OF ORGANIC ACIDS USING THE KROMASIL-CHI-TBB CHIRAL STATIONARY PHASE

Derivatives of racemic mandelic and tropic acids have been separated directly on a t-butylbenzoylated tartaric acid-based chiral stationary phase (CSP) (Kromasil-CHI-TBB) on both analytical and preparative scales. The resolution of the enantiomers of the model compound, rac-3-methoxytropic acid, was optimized by changing the nature and the composition of the mobile phase. The dominating interaction between the CSP and these hydroxy acids was found to be hydrogen-bonding. Consequently, retention of the analytes on the CSP decreased with increasing polarity of the mobile phase modifier. The separation of the enantiomers was strongly influenced by the π-donating and π-accepting ability of the aryl moiety, and strong electron-withdrawing substituents in the aryl group e.g.,—CF3 result in a loss of chiral recognition. However, the CSP can discriminate between the two enantiomers only when both the aryl moiety and the carboxylic function are directly bonded to the asymmetric carbon atom. The Kromasil-CHI-TBB column displays broad selectivity for the enantiomers of mandelic and tropic acid derivatives, which are relatively simple structures and not easily resolved. Even though the α-values are moderate, the high efficiency of the column facilitates the use of the CSP for the preparative scale chiral separation of a number of these compounds. Chirality 11:420–425, 1999.