Chiral assay methods

Walters, R.R. Hsu, C.Y.L. Chiral assay methods for lifibrol and metabolites in plasma and the observation of unidirectional chiral inversion following administration of the enantiomers to dogs. Chirality 1994, 6, 105-115. 

Determination of the drug substance is expected to be enantioselective, and this may be achieved by including a chiral assay in the specification or an achiral assay together with appropriate methods of controlling the enantiomeric impurity. For a drug product where racemization does not occur during manufacture or storage, an achiral assay may suffice. If racemization does happen, then a chiral assay should be used or an achiral method combined with a validated procedure to control the presence of the other enantiomer. 

Enantiomeric pnrity assays have also been performed without chromatographic separation being conducted prior to detection, for example, with circular dichroism (CD) and MS. Bertncci et al. [110] developed a chiral assay for pulegone, oxazepam, and warfarin by combining simnltaneons UV, CD, and g factor detection on an achiral separation system with a Hypersil CN colnmn and a mobile phase of hexane 2-PrOH (90 10). The precision (RSD%) of the method ranged from 0.6% to 2.6%, and the LOQs were between 0.1% and 1% (0.2-2.2 j,g). For fnrther information concerning the application of CD and polarometric detection for chiral detection, see the review by Bobbitt and Linder [111]. 

Description of special analytical method(s) for chiral assays... 

The Fab -functionalized nanotubes were added to racemic mixtures of the SR and RS enantiomers of FTB. The tubes were then collected by hltration and the filtrate was assayed for the presence of the two enantiomers using a chiral HPLC method (Figure 24.5B). Chromatogram I was obtained from a solution that was 20 pM in both enantiomers, and chromatogram II was obtained for the same solution after exposure to the Fab-functionalized nanotubes 75% of the RS enantiomer and none of the SR enantiomer was removed by the nanotubes. When the concentration of the racemic mixture was dropped to 10 pM, all the RS enantiomers were removed (chromatogram III). Nanotubes containing no Fab did not extract measurable quantities of either enantiomer from the 20 pM solution. 

The specifications for both enantiopure and racemic chiral drug substances should be sufficient to establish both chemical and stereochemical aspects of identity, strength, quality and purity. This implies both that the identity test use a stereochemically specific method and that the assay method be stereochemically selective. 

As with the bulk drug substance, spedfications for both enantiopure and racemic chiral drug products should include both a stereochemically spedfic identity test and stereochemically selective assay method. The analytical method to be used should not be arbitrarily chosen to be the same as for the bulk drug but should be chosen on the basis of the composition, method of manufacture, and stability characteristics of the formulation. 

The reported 100% optical purity for both of the alcohols 24 and 25 was based on NMR assay methods, so it may be prudent to assume a lower limit in the range of 95% ee. Nevertheless, it is clear that highly enriched products were obtained and separated. Furthermore, Davies and Jones were able to demonstrate an application to the synthesis of chiral secondary alcohols. Thus, desulfurization of 24 and 25 afforded enantiomerically pure 3-hexanol in good yield. 

The magnitude of the chemical shift non-equivalence is proportional to the size of the applied magnetic field. Lowering the temperature at which the spectrum is recorded can accentuate the anisochronicity between diastereomers. The use of non-polar solvents such as d-chloroform and, in particular, aromatic solvents such as de-benzene or dg-toluene offers considerable advantages. This effectively excludes the application of NMR methods for the assay of the enantiomeric purity of substrates which are only soluble in polar solvents like de-DMSO. It is unfortunate that numerous pharmacologically important compounds fall into this category. In such cases chiral GC or chiral HPLC methods may afford viable alternatives. Proton, i9p and 3ip are the most frequently studied nuclei. It is important to note that measured integrals will only report reliably on the enantiomeric purity in fully relaxed spectra free from any saturation effects. 

The guideline states that the objective of validation is to demonstrate that an analytical method is fit for its purpose and summarizes the characteristics required of tests for identification, control of impurities and assay procedures (Table 13-2). As such, it applies to chiral drug substances as to any other active ingredients. Requirements for other analytical procedures may be added in due course. 

Molecules which exhibit optical activity are molecules which have a handedness in their structure. They are chiral . Chemists often have reasons to obtain chemical pure aliquots of particular molecules. Since the chirality of molecules can influence biological effect in pharmaceuticals, the chiral purity of a drug substance can pose a challenge both in terms of obtaining the molecules and in assaying the chiral purity by instrumental methods. While diastereomers can have different physical properties including solubility, enantiomers have the same physical properties and the same chemical composition. How then to separate optically active molecules ... 

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

Amino acid enantiomers can be separated on a chiral stationary phase after derivatization with chloroformates (Abe et al., 1996). The derivatization procedure is quite simple and rapid, but the derivatizing reagent must be synthesized, which complicates the assay. Another method for the analysis of amino acid enantiomers uses N,0-pentafluoropropionyl isopropyl derivatives and a chiral column with NPD detection (Hashimoto et al., 1992). 

Chiral or achiral assay and purity determinations are done according to an external calibration calculation procedure, either with or without internal standardization. The calibration is performed against a 10% w/w (compared to the nominal concentration of the sample solution at 100% w/w) reference standard solution. The sample solution for the purity determination remains at the 100% w/w level, while that of the assay determination is diluted 10 times. The reason for the difference in concentration levels is similar to the purity method. A suggested sample injection sequence can be... 

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