Chiral AGP Research Articles

Pharmacokinetic behaviour of albendazole sulphoxide enantiomers in male and female sheep.

Benzimidazole anthelmintic drugs are widely used in veterinary practice. Albendazole sulphoxide (ABZSO) is a benzimidazole drug with two enantiomers, as a consequence of a chiral centre in the sulphoxide group. The kinetics of these enantiomers were studied in male and female sheep. Plasma samples were obtained from the animals between 0.5 and 72 h after oral administration of 7.5 mg/kg of a racemic formulation of ABZSO (total-ABZSO). After a liquid-liquid extraction, the samples were analysed by HPLC to determine the concentrations of total-ABZSO and of the sulphone metabolite (ABZSO2). During the chromatographic analysis, the ABZSO peak was collected and reanalysed by an HPLC technique using a Chiral AGP column to quantify the enantiomeric proportion therein. After kinetic analysis, the AUCs obtained for the (+)-ABZSO were 5.8 and 4.0 times higher than those for the (-)-ABZSO in male and female animals, respectively. The mean residence times were 23.4 and 16.1 h for (+)-ABZSO and 22.2 and 17.4 h for (-)-ABZSO for male and female animals, respectively. The only significant difference between the sexes (p < 0.05) was in the Tmax of the (-)-ABZSO. Comparing both enantiomers within each sex, significant differences were found in all the kinetic parameters. Finally, no kinetic differences were found between sex for total-ABZSO or ABZSO2.

Enantioselective high-performance liquid chromatographic method for the determination of methadone and its main metabolite in urine using an AGP and a C 8 column coupled serially

A simple and sensitive method for the enantioselective high-performance liquid chromatographic determination of methadone and its main metabolite, EDDP, in human urine is described. (−)-( R)-Methadone, (+)-( S)-methadone, (+)-( R)-EDDP, (−)-( S)-EDDP and imipramine as an internal standard are detected by ultraviolet detection at 200 nm. The enantiomers of methadone and EDDP were extracted from human urine by a simple liquid–liquid extraction procedure. The extracted sample was reconstructed in mobile phase and the enantiomers of methadone and EDDP were quantitatively separated by HPLC on a short analytical LiChrospher RP8 column coupled in series with a chiral AGP column. Determination of all four enantiomers was possible in the range of 0.03 to 2.5 μ M. The recoveries of methadone enantiomers and EDDP enantiomers added to human urine were about 90% and 80%, respectively. The method was applicable for determination of methadone enantiomers and the enantiomers of its main metabolite in urine samples from methadone maintenance patients and patients suffering from severe chronic pain.

Quantitative determination of albendazole and its main metabolites in plasma

Three different and complementary chromatographic methods are described for quantitative determination of albendazole (ABZ) and its two main metabolites: albendazole sulphoxide (SOABZ) and albendazole sulphone (SO 2ABZ). ABZ, SOABZ and SO 2ABZ can be quantified by two RP-HPLC methods with an ODS2 column and two different mobile phases. One of methanol–water (60:40) for ABZ and a second one of phosphoric acid in water–acetonitrile (80:20) for SOABZ and SO 2ABZ. SOABZ bears an asymmetric sulphur centre. Quantitative assay of (+) SOABZ and (−) SOABZ can be performed by HPLC. A chiral AGP column and a mobile phase of sodium phosphate buffer (8 m M, pH 7.0) containing different amounts of 2-propanol between 0 to 2% were used. Pharmacokinetic characteristics of ABZ following oral administration of a liquid formulation of ABZ (12 mg/kg) in mice has been studied with these three complementary HPLC methods and the results are reported.

Stereoselective analysis of benazepril and its stereoisomers by reversed-phase high-performance liquid chromatography on a chiral AGP column

A direct HPLC method was developed for the determination of benazepril, its stereoisomers and its active metabolite benazeprilat. The separation was achieved, without any derivatization, on an α 1-acid glycoprotein-based column (chiral AGP). The influence of pH, temperature, buffer ionic strength and organic modifiers of the mobile phase on retention and enantioselectivity was evaluated. The optimised method permits baseline separation of benazepril, its stereoisomers and its metabolite in less than 35 min, without the use of charged organic modifiers or tandem coupled chromatography.

Stereoselective determination of R,S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid and its taurine conjugates in dog urine by high-performance liquid chromatography

Two high-performance liquid chromatographic methods for the stereoselective determination of R,S-2-[4-(3-methyl-2-thienyl)-phenyl]propionic acid ( R,S-MTPPA), a new anti-inflammatory agent, and its taurine conjugates ( R,S-MTPPA-TAU) in dog urine have been developed and validated. The urine samples were subjected to solid extraction or TLC preparation, then R,S-MTPPA and R,S-MTPPA-TAU were separated on Chiralcel OD and Chiral AGP columns, respectively, with ultraviolet absorbance detection at 272 nm. The dose–response relationships for enantiomers of R,S-MTPPA and R,S-MTPPA-TAU were linear in the concentration ranges of 0.5–50 ( r>0.9993) and 5–200 μg/ml ( r>0.9982), respectively. Recoveries of all tested enantiomers from dog urine were roughly 90% within the above concentration ranges. Intra- and inter-day reproducibilities were sufficient for metabolic studies. These methods were applied to stereoselective determination of the enantiomers in dog urine after administration of either S- or R-MTPPA.

Kinetics of racemization of (+)- and (-)-diethylpropion: studies in aqueous solution, with and without the addition of cyclodextrins, in organic solvents and in human plasma.

The configurational stability of (+)- and (-)-diethylpropion [(+)- and (-)-2-(diethyl)-1-phenyl-1-propanone or (+)- and (-)-DEP] was investigated systematically from chemical, pharmaceutical, and pharmacological aspects. The enantiomeric ratio was monitored directly with a recently developed stability-indicating enantioselective HPLC method. In aqueous solutions, the rate of racemization increased non-linearly with increasing pH and with increasing phosphate buffer concentration. The racemization rate showed a positive slope with increasing temperature and decreasing ionic strength. The racemization rates of (+)- and (-)-DEP in the presence of cyclodextrins (CDs) did not differ significantly. CDs that were added to (+)- and (-)-DEP in a molar ratio 5:1 showed the following effects after dissolution in 10 mM phosphate buffer (final pH 6.7): sulfobutyl ether-beta-CD (SBE-beta-CD) and methylated-beta-CD (Me-beta-CD) retarded racemization; whereas hydroxypropyl-beta-CD (HP-beta-CD), acetyl-gamma-CD (Ac-gamma-CD), acetyl-beta-CD (Ac-beta-CD), gamma-CD, and beta-CD showed a weak destabilising effect. In contrast to the described CDs, alpha-CD distinctly accelerated the rate of racemization. The configurational stability of (+)- and (-)-DEP was also studied under physiological conditions. The half-life of racemization in heparinised human plasma was for both enantiomers determined to be approximately 23-25 min. In phosphate buffer (10 mM, pH 7.4), rac-DEP showed a high, but unselective affinity towards human alpha 1-acid glycoprotein (orosomucoid) immobilised on silica (Chiral AGP). The rate of racemization of the free base of (-)-DEP dissolved in organic solutions generally increases with the polarity of the solvating agent.

Kinetics of racemization of (+)‐ and (−)‐diethylpropion: Studies in aqueous solution, with and without the addition of cyclodextrins, in organic solvents and in human plasma

The configurational stability of (+)- and (−)-diethylpropion [(+)- and (−)-2-(diethyl)-1-phenyl-1-propanone or (+)- and (−)-DEP] was investigated systematically from chemical, pharmaceutical, and pharmacological aspects. The enantiomeric ratio was monitored directly with a recently developed stability-indicating enantioselective HPLC method. In aqueous solutions, the rate of racemization increased non-linearly with increasing pH and with increasing phosphate buffer concentration. The racemization rate showed a positive slope with increasing temperature and decreasing ionic strength. The racemization rates of (+)- and (−)-DEP in the presence of cyclodextrins (CDs) did not differ significantly. CDs that were added to (+)- and (−)-DEP in a molar ratio 5:1 showed the following effects after dissolution in 10 mM phosphate buffer (final pH 6.7): sulfobutyl ether-β-CD (SBE-β-CD) and methylated-β-CD (Me-β-CD) retarded racemization; whereas hydroxypropyl-β-CD (HP-β-CD), acetyl-γ-CD (Ac-γ-CD), acetyl-β-CD (Ac-β-CD), γ-CD, and β-CD showed a weak destabilising effect. In contrast to the described CDs, α-CD distinctly accelerated the rate of racemization. The configurational stability of (+)- and (−)-DEP was also studied under physiological conditions. The half-life of racemization in heparinised human plasma was for both enantiomers determined to be approximately 23–25 min. In phosphate buffer (10 mM, pH 7.4), rac-DEP showed a high, but unselective affinity towards human α1-acid glycoprotein (orosomucoid) immobilised on silica (Chiral AGP). The rate of racemization of the free base of (−)-DEP dissolved in organic solutions generally increases with the polarity of the solvating agent. Chirality 10:307–315, 1998. © 1998 Wiley-Liss, Inc.

Direct liquid chromatographic enantioseparation of sotalol and other β-blockers using an α1-acid glycoprotein-based chiral stationary phase

The behaviour of an α 1-acid glycoprotein-based chiral stationary phase (Chiral AGP) towards changes in pH and organic modifier in the mobile phase was investigated in order to deduce suitable conditions for the liquid chromatographic enantioseparation of a series of β-adrenoreceptor blocking drugs. The effects of the pH of the mobile phase on retention, selectivity and resolution were studied. Methanol was the only non-ionic modifier tested in the mobile phase while different aliphatic carboxylic acids (C 4 to C 8) and alkanesulfonic acids (C 6 to C 8) were used as ionic modifiers. The influence of the nature and concentration of these modifiers on retention and enantioselectivity was investigated. Under these conditions, enantiomeric separations could be obtained for more than 70% of the β-blocking agents examined. The use of heptanoic acid as an ionic additive in the mobile phase has permitted the resolution of sotalol enantiomers. An enantioselective assay for sotalol was then developed and validated.

Comparison of capillary electrophoresis and liquid chromatography for the enantiomeric separation of ?-phosphonosulfonic acids

The enantiomers of α-phosphonosulfonic acids were completely resolved by capillary electrophoresis using β-cyclodextrin as a chiral selector in a borate electrolyte and HPLC using a chiral AGP column. The methods were used to quantitate the R-enantiomer present as an impurity in the S-enantiomer, a potential cardiovascular drug candiate. Chirality 9:104–108, 1997. © 1997 Wiley-Liss, Inc.

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester.

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 micron silica-bonded, alpha 1-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1-4% (r > 0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50 degrees C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r > 0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.

Separation of Rosmarinic Acid Enantiomers by Three Different Chromatographic Methods (HPLC, CE, GC) and the Determination of Rosmarinic Acid inHedera helix L.

Three methods employing high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography (GC) were developed for the separation of enantiomers of rosmarinic acid (RA). Chiral resolution of underivatized RA was achieved on a chiral AGP® HPLC column and by micellar electrokinetic capillary chromatography (MECC). Alternatively, after derivatization, the resulting diastereomeric 3,4-di-hydroxyphenyllactic acid (-)-menthyl esters were separated by non-chiral GC. These methods allow reliable and rapid determination of the enantiomeric ratio of RA samples. RA obtained from vegetative material of Hedera helix L. (Araliaceae) was determined to be (R)-(+)-RA. In contrast to published data, the optical rotation value of rosmarinic acid was found to be +106°.

Determination of ketamine and norketamine enantiomers in plasma by solid-phase extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method is described for determination of sub-anaesthetic concentrations of the enantiomers of ketamine and its metabolite norketamine in plasma. The samples are purified by reversed-phase solid-phase extraction. The enantiomers are separated on a Chiral AGP column with a mobile phase containing 16% methanol and a 10 m M phosphate buffer at pH 7.0, and measured by UV-detection at a wavelength of 220 nm. Linear calibration curves with correlation coefficients better than 0.995 have been obtained in the range 10–320 ng/ml. Minimum detectable concentrations were about 2 ng/ml.

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes.

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantio-selectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other.

Enantioselective Determination of Mianserin and Its Desmethyl Metabolite in Plasma During Treatment of Depressed Japanese Patients

A method for the analysis of the enantiomers of mianserin and its main metabolite desmethylmianserin in plasma using a chiral AGP high performance liquid chromatography column is described. In the majority of 66 depressed Japanese patients, the concentration of the more active S(+)-mianserin was higher than that of the R(-)-enantiomer. The mean S/R ratio was 1.9 +/- 0.9 (SD) (range 0.5-4.8). The concentration of S(+)-varied between 19 and 223 nM, while that of the R(-)-mianserin varied between 14 and 72 nM. Among 42 of the 66 plasma samples in which the enantiomers of desmethylmianserin could be measured, the R-form predominated in all but one. S-Desmethylmianserin was in most of the samples present in concentrations of < 10 nM (limit of detection). Analysis of the enantiomers of mianserin and desmethylmianserin might allow the establishment of a closer relationship between plasma levels and clinical effects.

Chiral separation of MDL 73,005EF enantiomers using an alpha 1-acid glycoprotein column.

The enantiomeric separation of MDL 73,005EF (racemic mixture of two enantiomers) has been accomplished using a Chiral AGP (alpha 1-acid glycoprotein) column. The enantiomers are baseline resolved with a runtime of less than 10 min. This separation is used to quantitate the enantiomers present in bulk drug samples, tabletted formulations, and drug used in pharmacological and toxicological studies. Variables found to have an effect on the enantiomeric separation were studied and include: mobile phase ionic strength, type and concentration of organic modifier added to the mobile phase, mobile phase, mobile phase pH, column temperature and the amount of analyte injected. The enantiomeric separation was optimized on the Chiral AGP column based on the effects that each variable had on the separation. Calibration curves for a standard were linear over a range of 0.24 to 61.2 micrograms/g with a correlation coefficient of better than 0.999. A detection limit of 0.012 micrograms/g and a quantitation limit of 0.24 micrograms/g were also found.

Determination of the stereoselective aspects in in-vitro and in-vivo metabolism of the analgesic meptazinol by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method to separate meptazinol and its phase I metabolites has been developed using a LiChrosper 100 CN column and a mobile phase of trimethylammoniumacetate buffer (pH 5.5)-acetonitrile-methanol. Quantification of meptazinol and N-desmethylmeptazinol in biological samples was achieved by extraction with organic solvents and chromatographic analysis (detection limit 0.4 and 0.25 micrograms/ml, respectively). Afterwards the enantiomeric ratio of the two compounds was determined on a Chiral AGP column with a mobile phase of phosphate buffer (pH 7.0)-acetonitrile (alpha = 1.29 and 1.49, respectively). In-vitro metabolism data after incubation of the racemic compound and the enantiomers with liver supernatant and microsomes of different species are presented. Finally urinary data of two volunteers after oral application of the racemic drug were determined.

Enantioselective high-performance liquid chromatographic method for the determination of methadone in serum using an AGP and a CN column as chiral and analytical column, respectively

A simple and sensitive HPLC method with ultraviolet absorption detection at 200 nm is described for the determination of methadone enantiomers in human serum, using dextropropoxyphene as an internal standard and organic solvent extraction. Separation was performed on two serially coupled columns, CN and Chiral AGP, with a mobile phase consisting of acetonitrile, dimethyloctylamine and phosphate buffer. Using 1.0 ml of serum, 5 nmol/1 of each enantiomer could be determined with an acceptable precision. No interactions from several drugs were observed. The method has been successfully used in a pharmacokinetic study. More than 2500 serum samples have been separated on the same AGP colunm with acceptable selectivity and resolution.

Isomeric separation of Beraprost sodium using an α 1-acid glycoprotein column

The separation of the four stereoisomers present in Beraprost sodium, a prostacyclin analogue, has been accomplished using an α 1-acid glycoprotein stationary phase (Chiral AGP column). The stereoisomers are baseline resolved with a runtime of less than ten minutes. This chiral separation is used to quantitate the four Beraprost sodium stereoisomers present in the bulk drug, tabletted formulations and in pharmacological and toxicological studies. The mobile phase variables that were found to have an effect on the stereoisomeric separation were studied and include: ionic strength, type and concentration of organic modifier, mobile phase pH and column temperature. Optimum stereoisomer separation was achieved on the Chiral AGP column. Calibration curves were linear for all four stereoisomers over the range of 0.024 to 4.04 μg/ml using fluorescence detection (correlation coefficients were greater than 0.999). A detection limit of 0.004 μg/ml was found for each stereoisomer. This assay has been used to determine the ratio of the four stereoisomers in the bulk drug as well as in the final formulated tablets.

Enantiomeric LC separation of calcium antagonists on protein‐based chiral stationary phases

AbstractThree chiral calcium antagonist drugs, bepridil and two dihydropyridine derivatives (nicardipine and REC 15/2375), have been successfully separated within short retention times using either the α1‐acid glycoprotein chiral stationary phase (Chiral AGP) or the ovomucoid column (Ultron ES‐OVM). Aqueous buffer at defined pH is modified by the addition of an organic component (propan‐2‐ol, acetonitrile, ethanol) in order to modulate the retention properties of each system. The influence of pH and percentage of organic modifier on retention, selectivity, resolution, and column performance are discussed for bepridil analyzed on Chiral AGP and for the two dihydropyridines (nicardipine and REC 15/2375) analyzed on Ultron ES‐OVM stationary phases. © 1993 Wiley‐Liss, Inc.

Clinical Pharmacokinetics of R(+)-and S(???)-Mepivacaine After High Doses of Racemic Mepivacaine With Epinephrine in the Combined Psoas Compartment/Sciatic Nerve Block

The purpose of this study was to investigate the pharmacokinetics of R(+)- and S(-)-mepivacaine in 10 male patients after injection of a high dose (731 mg) of racemic R,S-mepivacaine for a combined psoas compartment/sciatic nerve block. Arterial blood samples were taken, and the plasma concentrations of the stereoisomers R(+)- and S(-)-mepivacaine were measured by means of high-performance liquid chromatography (HPLC) with a Chiral AGP column. The S(-) isomer reached higher plasma concentrations than the R(+) isomer. The maximal plasma concentration (Cmax) of R(+) was 1.54 +/- 0.34 micrograms/mL, whereas that of the S(-) isomer was 2.34 +/- 0.51 micrograms/mL (P = 0.00050). The time at which Cmax was reached (Tmax) was identical for both isomers. The area under the plasma concentration-time curve from t = 0 to infinity (AUC infinity) of S(-)-mepivacaine was almost double that of R(+)-mepivacaine. The elimination half-life (T1/2) was identical for both isomers (3 h), which means that the calculated total body clearance and the calculated steady-state volume of the distribution of R(+) are, respectively, 1.7 and 1.5 times larger than that of the S(-) isomer. We conclude that the plasma concentrations of the S(-)-mepivacaine isomer were higher than those of the R(+) isomer because of a smaller volume of distribution and a slower total body clearance.