Chromatographic Methods Using Chiral Columns

A liquid chromatographic method is utilized for the determination of clopidogrel bisulfate in samples of the bulk drug substance. The method uses a column (L57 column size 15 cm x 4.6 mm) packed with ovomucoid (a chiral-recognition protein) that is chemically bonded to silica particles of 5 /im diameter and a pore size of 120 A. Both the reference standard and the sample to be analyzed are dissolved in methanol, and then diluted with mobile phase. The mobile phase is 75 25 0.01 M phosphate buffer /acetonitrile, and the flow rate is adjusted to 1.0 ml/min. Observation is made on the basis of the UV absorbance at 220 nm, and the clopidogrel peak has a relative retention time about 1.0 min. 

Each glycopeptide CSP has unique selectivity as well as complementary characteristics, and a considerable number of racemates have been resolved on all three of them. Interestingly, most of the resolved enantiomers have the same retention order on these macrocyclic CSPs. When they are mixed or coupled with each other, the selectivity on one CSP will not be canceled by another. Even if some compounds may not have the same retention order, the complementary effects will result in an identifiable selectivity. Therefore, the coupled chiral columns can be used as a screening tool and save chromatographers substantial time in method development. 

Mass spectrometry (MS) is also being used to add another dimension of analysis to achiral-chiral analysis. Recently, an achiral-chiral column-switching LC/LC-MS/MS method was reported for the pindolol enantiomers in human serum (Motoyama et al., 2002) and phenprocoumon metabolites (Kammerer et al., 1998). For analytes that have very poor chromophores or cannot naturally fluoresce, MS detection can be more sensitive for the underivatized form of the analyte. Also, MS detection can be particularly useful when very similar analytes that differ in mass (such as some amino acids and metabolites) cannot be satisfactorily separated chromatographically,... 

Enantiomeric purity. In order to assess the efficiency of an enantioselective hydrolase-catalyzed reaction, it is imperative that one can accurately measure at least the conversion and the enantiomeric excesses of either the substrate or the product (see equations Equation 1, Equation 2, and Equation 3). Although optical rotation is sometimes used to assess enantiomeric excess, it is not recommended. Much better alternatives are various chromatographic methods. For volatile compounds, capillary gas chromatography on a chiral liquid phase is probably the most convenient method. Numerous commercial suppliers offer a large variety of columns with different chiral liquid phases. Hence it is often easy to find suitable conditions for enantioselective GC-separations that yield ee-values in excess of... 

Martens-Lobenhoffer et al. [119] used chiral HPLC-atmospheric pressure photoionization tandem mass-spectrometric method for the enantio-selective quantification of omeprazole and its main metabolites in human serum. The method features solid-phase separation, normal phase chiral HPLC separation, and atmospheric pressure photoionization tandem mass spectrometry. The internal standards serve stable isotope labeled omeprazole and 5-hydroxy omeprazole. The HPLC part consists of Agilent 1100 system comprising a binary pump, an autosampler, a thermo-stated column component, and a diode array UV-VIS detector. The enantioselective chromatographic separation took place on a ReproSil Chiral-CA 5 ym 25 cm x 2 mm column, protected by a security guard system, equipped with a 4 mm x 2-mm silica filter insert. The analytes were detected by a Thermo Scientific TSQ Discovery Max triple quadrupole mass spectrometer, equipped with an APPI ion source with a... 

Diastereomeric complexes can also be formed by ion-pairing of an enantiomer with a chiral counterion. In order to form this diastereomeric complex, it has been postulated that at least three interaction points between the ion pair are required [250]. Nearly all of these form weak complexes in aqueous mobile phases. Consequently, the chromatographic methods that have been developed have been either silica or diol columns with low-polarity mobile phases. Enantiomeric amines, such as the beta-blockers, have been optically resolved when (-l-)-lO-camphorsulfonic acid was used as the chiral counterion [251]. Enantiomers of norephedrine, ephedrine, pseudoephedrine, and phenyramidol have all been resolved from their respective enantiomers with n-dibutyltartrate [252]. Enantiomers of naproxen, a chiral carboxylic acid, are resolved from each other by either using quinidine or quinine in the mobile phase [253]. In these studies, silica... 

A simple, isocratic chromatographic method for the separation, identification, and measurement of etodolac enantiomers without derivitization using chiral stationary phase columns has been reported [25]. A chiral stationary phase column packed with Chiracel OD (cellulose tris-3,5-dimethylphenylcarbamate coated on 10 pm silica gel) was used as the stationary phase. The mobile phase (85 15 v/v, n-hexane/2-propanol (containing 0.1% trifluoroacetic acid)) was pumped at 0.7 mL/min and the UV detection was set at 230 nm. The (-)-(/ )-etodolac enantiomer eluted first, indicating its stronger interaction between the stationary phase relative to the (+)-(S)-etodolac enantiomer. 

A micellar electrokinetic chromatographic method allows the separation of optically isomeric diltiazem hydrochloride using bile salts as chiral surfactants. The chiral separation of diltiazem hydrochloride from several analogs is achieved at ambient temperature using 0.05M sodium taurodeoxycholate in a 0.02M aqueous phosphate-borate buffer solution of pH 7.0. Separation is performed using a fused-silica capillary tube (650 mm x 50 mm I.D.) and a voltage up to +25 kV. Detection is achieved on-column using UV adsorption at 210 nm (31). 

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