Direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using a chiral alpha 1-acid glycoprotein column

A 10-cm long alpha 1-acid glycoprotein column is used for the enantiomeric resolution of the clinically used racemic aminoglutethimide (+/- AG) and its acetylated metabolite (+/- AAG). A direct liquid chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite is accomplished without any derivatizations. Maximum resolutions of 1.37 and 0.73 are obtained for the enantiomers of aminoglutethimide and its acetylated metabolite, respectively. The effect of the 2-propanol content in mobile phase on retention and enantioselectivity of aminoglutethimide and its acetylated metabolite is demonstrated. The variation of the separation factors (alpha) with pH in enantiomeric separation of aminoglutethimide is also shown.     

Liquid chromatographic determination of the enantiomers of ibuprofen in plasma using a chiral AGP column

A reversed-phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen. The enantiomers and the internal standard 4-pentylphenylacetic acid are extracted from plasma, separated and quantified on a Chiral-AGP column using ultraviolet detection. The simplicity, sensitivity and precision of the method makes it convenient for use in pharmacokinetic studies.     

Enantiospecific high-performance liquid chromatographic analysis of 2-phenylpropionic acid, ketoprofen and fenoprofen

A high-performance liquid chromatographic method has been developed for the quantitation of the R- and S-enantiomers of 2-phenylpropionic acid, ketoprofen and fenoprofen. The assay consists of extracting the arylpropionic acid with an internal standard and measuring the total (R + S) concentration of enantiomers by reversed-phase chromatography, derivatising the chromatographic fraction corresponding to the enantiomers to form R- and S, R-2-phenylethylamide distereoisomers which are resolved by normal-phase chromatography in order to calculate the fraction of each enantiomer. The limits of sensitivity of the assay for 2-phenylpropionic acid, ketoprofen and fenoprofen are 6, 0.2 and 2.5 mg/l, respectively.     

Rapid and simple method for the determination of alpha 1-acid glycoprotein in serum by column liquid chromatography.

A rapid and simple method for the determination of alpha 1-acid glycoprotein (AAG) in serum was developed by using an anion-exchange column for clean-up of serum and a hydroxyapatite column for high-performance liquid chromatography (HPLC). A good correlation was observed between this HPLC method and the conventional radial immunodiffusion method. The method may also be used to determine the AAG concentration in the serum of experimental animals.     

Chiral high-performance liquid chromatographic separations of vinca alkaloid analogues on alpha 1-acid glycoprotein and human serum albumin columns.

Separations of the stereoisomers of a series of tetracyclic and pentacyclic vinca alkaloid analogues having two or three chiral centres were performed on Chiral-AGP and Chiral-HSA high-performance liquid chromatographic columns. Phosphate buffers with pH 5-7 containing 5-35% acetonitrile or 2-propanol were used as mobile phases. The results were in accordance with previous binding data obtained with native AGP and on an HSA-Sepharose column. Whereas on Chiral-AGP the retention of the trans isomers having 1(R),12b(S)-indolo[2,3-a]quinolizidine or the corresponding 3(S),16(R)-eburnane absolute configurations was exceedingly high, on Chiral-HSA the trans isomers, independently of their absolute configurations, were more retained. Eburnane-type compounds could also be separated according to the configuration of the chiral centre at position 14. A comparison of the chromatographic properties of the vinca alkaloids on the Chiral-AGP and Chiral-HSA columns demonstrates that these compounds are bound with higher affinity to the AGP phase. The AGP column resolves a very broad range of vinca alkaloids compared with the HSA column. Higher stereoselectivity and a much better chromatographic performance were also obtained on the Chiral-AGP column.     

Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma.

An enantioselective high-performance liquid chromatographic assay for the quantitation of the enantiomers of ketamine and its major metabolite norketamine in human plasma is described (assay I). The procedure involved extraction of the compounds from alkalized plasma into cyclohexane. Stereoselective separation was achieved with a prepacked alpha 1-acid glycoprotein column without any derivatization procedure. A second assay using a conventional reversed-phase column to determine total (racemic) ketamine and norketamine is also described. Because of interfering plasma peaks (assay II) the cyclohexane solution was reextracted into 1 M hydrochloric acid. The detection wavelength was 215 nm for all substances. The limit of quantification of the method was ca. 40 ng/ml in plasma. The assays were sensitive and reproducible. The method was demonstrated to be sensitive for stereoselective pharmacokinetic studies of ketamine after clinical doses.     

Relationship between enantioselectivity and solute structure on a chiral α1-acid glycoprotein column

Summary The relationship between the enantioselectivity, the retention and the solute structure for N-aminoalkylsuccinimides and 1-alkyl-2′,6′-pipecoloxylidides have been studied using α₁-acid glycoprotein as the chiral stationary phase. Both the enantioselectivity and the retention are highly affected by small changes of the solute structure in series of homologous compounds. For example, increasing the steric bulk on a basic aliphatic nitrogen improves the enantioselectivity. It was also demonstrated for the succinimides that the magnitude of the separation factor was highly affected by the distance between the basic nitrogen and the chiral center. The chiral selectivity was not lost for the succinimides despite the fact that the basic nitrogen was separated from the chiral center with seven atoms, which may be an effect of that these compounds contain more than one strong hydrogen bonding group. Substitution in the aromatic ring of the succinimides also influences both the retention and the chiral selectivity to a large extent. Electronegative substituents can increase the separation factor but introduction of an alkyl or an alkoxy group in the aromatic ring decreases the enantioselectivity for the succinimides.     

Isocratic high-performance liquid chromatographic determination of plasma adenosine

Summary Due to manifold physiological and cardioprotective actions of adenosine, the demand for a simple but accurate method to determine its concentration in plasma is increasing. The aim of this study was firstly to develop a simple isocratic method instead of the gradient elution or peak-shifting techniques used earlier and secondly to check conflicting data on the composition of stop-solution, added to the sample in order to prevent changes in adenosine concentration. Isocratic elution improved signal to noise ratio and concentrations of 100 mol L^–1 dipyridamole and 2.5 mol L^–1 erythro-9(2-hydroxy-3-nonyl)adenine in the blood sample effectively prevented both adenosine formation and degradation, even without the use of a 5-ecto-nucleotidase inhibitor. Lowering the concentration of dipyridamole to 25 mol L^–1 caused more than a tenfold increase of adenosine concentration in two out of five cases and even 100 mol L^–1 dipyridamole alone is not sufficient to inhibit adenosine deaminase in blood samples.     

Alpha 1-acid glycoprotein high-performance liquid chromatography column (EnantioPAC) as a screening tool for protein binding

An attempt was made to correlate retention behavior on a high-performance liquid chromatographic (HPLC) system employing an immobilized alpha 1-acid glycoprotein (AAG) column with AAG binding behavior for various compounds. Protein binding was assessed by propranolol displacement studies in an equilibrium dialysis system using isolated AAG. HPLC retention behavior poorly correlated with propranolol displacement from AAG. This system is not suitable for use as a screening tool for AAG affinity.     

Sensitive high-performance liquid chromatographic determination of pirenzepine in plasma

A high-performance liquid chromatographic method for the determination of pirenzepine in human plasma is reported using imipramine as an internal standard. The assay has a lower limit of detection of 2.5 ng/ml. The calibration function is found to be linear in the range from 5 ng/ml up to at least 100 ng/ml. Two sets of chromatographic conditions are described, which provide different chromatographic selectivities for the separation of the compounds of interest from other material present in a sample.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography.

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Stereoselective high-performance liquid chromatographic assay for pirmenol enantiomers in dog plasma.

Pirmenol enantiomers in dog plasma were quantified using a stereospecific high-performance liquid chromatographic method with ultraviolet detection at 262 nm. Racemic pirmenol and internal standard, (+)-propranolol, were isolated from dog plasma by a three-step extraction procedure using toluene, 0.1 M hydrochloric acid and hexane, respectively. A chiral analytical column (Chiralcel OJ) was used with a mobile phase consisting of hexane-isopropanol-diethylamine (98.9:1.0:0.1). Linear calibration curves were obtained in the concentration range 0.0200-5.00 micrograms/ml for each enantiomer. Precision of the method, expressed as coefficient of variation for nine quality control samples, was 7.1% for (+)-pirmenol and 6.4% for (-)-pirmenol. Bias was +/- 2.2% for (+)-pirmenol and +/- 1.5% for (-)-pirmenol in quality control samples.     

Investigation of chiral recognition mechanism on chicken alpha(1)-acid glycoprotein using separation system

Chicken alpha1-acid glycoprotein (alpha1-AGP) consists of 183 amino acid residues and has only one Trp residue at the 26 position. In this study, the Trp26 residue was modified with 2-nitrophenylsulfenyl chloride and chiral separation of neutral, acidic and basic compounds was examined on chicken alpha1-AGP and Trp-modified chicken alpha1-AGP columns. Chiral separation of propranolol, alprenolol and oxprenolol was lost on the Trp-modified chicken alpha1-AGP column, while chlorpheniramine, ketoprofen and benzoin were still enantioseparated on the Trp-modified chicken alpha1-AGP column despite of lower enantioselectivity than that on the chicken alpha1-AGP column. These results suggest that the Trp26 residue could be responsible for chiral recognition of these compounds. Competition studies using N,N-dimethyl-n-octylamine (DMOA) as a competitor indicated that propranolol, alprenolol and oxprenolol competed with DMOA on a single binding site near the Trp26 region and that further bindings of chlorpheniramine, ketoprofen and benzoin occurred at the secondary binding site in a non-competitive fashion with DMOA.