Analysis enantioseparation

Kappe CO, Shishkin OV, Uray G, Verdino P (2000) X-ray structure, conformational analysis, enantioseparation, and determination of absolute configuration of the mitotic kinesin Eg5 inhibitor Monastrol. Tetrahedron 56 1859-1862... 

Barretta and coworkers63 reported a direct determination of the enantiomeric purity of chiral trisubstituted allenes by using permethylated cyclodextrin as a chiral solvating agent. They found that the heptakis ft-cyclodextrin TRIMEB discussed above can be successfully used as a chiral solvating agent (CSA) for the NMR determination of the enantiomeric purity of trisubstituted allenes llOa-f. An accurate analysis of the experimental conditions (molar ratio aliene/TRIMEB, temperature and solvent) required to optimize the enantioseparation has been carried out. The XH NMR spectra of TRIMEB, allenes llOa-f, and the mixtures TRIMEB/allene have been recorded at 300 MHz in CD3OD as solvent. 

Some special approaches defined in the CE enantioseparation field can be found in References 37 and 38. In Reference 37, the authors tested four approaches to decrease the analysis time of a previously developed chiral separation method for amphetamine and its related compounds. The considered possibilities were (i) the short-end injection technique or (ii) increased electrical field combined with a capillary length reduction. 

Chip-based enantioseparations using an electrophoresis principle were presented by Gao et al. [59]. They used mono-, two-, and four-channel chips to develop chiral separations of fluorescein isothiocyanate (FlTC)-labeled basic compounds. To obtain the chiral separations, seven neutral CD were screened (i.e., a-CD, fi-CD, y-CD, HP-a-CD, HP-y-CD, and DM-fi-CD). Using the monochannel chip, the seven selectors were screened sequentially. Using the two-channel chip, between-channel repeatability could be demonstrated using the same separation conditions. Using two different selectors in the channels, the analysis time for the screening of the seven CD can be reduced to half, compared to the time needed in the monochannel... 

Note that not all enantioseparations in SFC are better than in HPLC [34], Bernal et al. [62] described the enantiomeric separation of several pharmaceutical-related compounds on a polysaccharide-based column using HPLC and SFC. They showed that most of the separations obtained by SFC are better, in terms of resolution and analysis time, than the separations obtained by HPLC. However, one compound could not be resolved using SFC, but LC provided baseline resolution. 

The use of polysaccharide-based CSPs instead of protein-based CSPs often increases the peak efficiency and facilifafes faster separafions. Papini ef al. [159] recently developed a method for the enantioseparation of lorazepam and on a Chiralpak OD-R column and an enzymatic hydrolysis was used to determine the amount of the glucoronide metabolite of lorazepam present. The separation was performed in 7 min with an LOQ of 1 and 10 ng/mL for lorazepam in plasma and urine, respectively. Another relatively fast separation for chiral analysis was published by Lausecker and Eischer [188]. They developed a method for determination of the drug candidate R483 within... 

The progress toward enantiomerically pnre drngs makes the selective and rapid analysis of enantiomers an important issue, both for chiral parity determinations and for enantioselective bioanalysis. Chankvetadze et al. [198] performed enantioseparations within an analysis time of 1 min for each of two chiral compounds (1,2,2,2-tetraphenylethanol and 2,2 -dihydroxy-6,6 -dimethylbiphenyl) by nsing a homemade capillary column containing monolithic silica modified with amylose tris(3,5-dimethylphenylcarbamate) (Figure 17.10). 

The analysis time for chiral HPLC separations will probably remain relatively long until CSPs with higher efficiency than the present ones become available. But monolithic columns, columns with a smaller particle size (i.e., UPLC ), and miniaturized systems would increase the efficiency and speed up the enantioseparation of existing types of CSPs. 

Recently, Yekkala et al. [144,145] have proposed an HPLC method with fluorescence detection. The method involves a rather laborious sample preparation due to the peculiar nature of the substrate involved (teeth), including pulverization, demineralization, hydrolysis of dentin and derivati-zation with o-phthaldialdehyde and A-acetyl-L-cysteine in order to obtain the enantioseparation of aspartic acid. Using a similar procedure, Benesova et al. [146] found that, in comparison with GC, HPLC provides shorter analysis time and higher sensitivity. 

Hence, many variables must be considered when optimizing a separation with respect to analysis time and resolution of enantioseparations. A natural extension was to develop computer programs to assess these variables and optimize the process automatically, and some general examples have been reported84,86. Numerous examples in addition to those in Table 2 of... 

Cass et al. [71] described a direct injection HPLC method, with column-switching, for the determination of omeprazole enantiomers in human plasma. A restricted access media of bovine serum albumin octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the restricted access media column onto an amylose tris (3,5-dimethylphenylcarbamate) chiral column by the use of a columnswitching valve and the enantioseparation was performed using acetonitrile-water (60 40) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40-mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. 

The enantioseparation represents a unique and very important field of separation science and is more and more frequently used in the analysis and purification of potential drugs. 

Because solutes have higher diffusion coefficients in super(sub)critical fluids than in liquids, the optimum linear velocity is shifted to higher values. Consequently, higher flow rates can be used leading to reduced analysis (separation) time without compromising efficiency. In addition, although the chiral discrimination ability of CSPs and enan-tioselectivity in SFC resemble usually those of non-aqueous LC, in some cases enantioseparations can be obtained in SFC which cannot be achieved in conventional LC. 

CEC was demonstrated in the analysis of /i-adrenergic antagonists using capillaries modified with propranolol-imprinted polymer. In situ molecular imprinting was performed via photo-polymerisation at — 20°C within a capillary that was premodified with 3-methacryloxypropyltrimethoxysilane. The time for UV irradiation was carefully determined so as to obtain a polymer coating of appropriate thickness. Enantioseparation of the racemate of propranolol was successfully demonstrated with a separation factor of 1.12 and a resolution factor of 1.26. 

Successful enantioseparation of individual N -protected amino acids stimulated the development of a rapid method of their simultaneous enantioseparation and quantification in a mixture. A feasibility study on this topic has been recently published by Welsch et al. [69]. The two-dimensional HPLC method involves online coupling of a narrow-bore C18 reverse phase (RP) column in the first dimension (separation of racemic amino acids) to a short enantioselective column based on nonporous 1.5 pm particles modified with t-BuCQD in the second dimension (determination of enantiomer composition). Using narrow-bore column resulted in fast analysis time for example, the mixture of nine racemic N-DNB-protected amino acids was completely analyzed within 16 min. 

Capillary electrophoresis and its most popular hybrid technique - capillary electrochromatography - are complementary to HPLC, offering rapid analysis, low consumption of sample and solvents, and usually a higher efficiency of separation (due to a larger number of theoretical plates). Similar to HPLC, enantioseparation with the use of electrophoretic methods can be conducted by direct (chiral phase... 

In this context, the on-hne coupling of CE with MS has evolved into an efficient, sensitive and selective technique for the chiral analysis of drugs. This chapter does not intend to cover aU the recent developments in the application of electromigration approaches coupled with conventional detection techniques such as UV, electrochemical, fluorescence or LIE [1, 2] for the enantioselective analysis of drugs. Moreover, achiral CE-MS apphcations have been summarised in recent reviews and these can be consulted for a more systematic coverage of the field [3-5]. This chapter will therefore focus on the electrophoresis-based enantioseparation technique coupled with mass spectrometry. 

The obvious advantages of a reduction of the column diameter in analytical enantioseparations include less consumption of packing materials and of high-purity organic solvents, less environmental problems, smaller sample size, and improved analytical characteristics (less dilution of sample, higher plate number and shorter analysis time) of the separation [120]. 

Direct HPLC enantioseparation techniques, which are free of many disadvantages of GC, indirect and chiral mobile phase HPLC methods, have gained unequivocal prevalence in bio-analytical studies. Several methods have been advanced so much that they allow enantiose-lective determination not only of the parent chiral drugs but also of their pharmacologically relevant metabolites [121]. As already mentioned above, a direct injection of biofluids offers several advantages in terms of analysis time and sample recovery. Precolumns packed with achiral or chiral packings, or with the recently developed so-called restricted-access packing materials, may be useful in this case. 

From their QSERR they find solute lipophilicity and steric properties as being responsible for analyte retention (k ) while enantioseparation (a) varied mainly with electronic and steric properties. The main difference between the analytes is that the enantioseparation of the esters is correlated with steric parameters that scale linearly with log a while the sulfoxides scale nonlinearly (parabolic), but this may be due to a computational artifact. The 3D-QSERR derived from field analysis revealed that while superpositioning of field maps for both analytes are not exactly the same, a similar balance of physicochemical forces involved in the chiral recognition process are at play for both sets of analyes. This type of atomistic molecular modeling, then, is a powerful adjunct to the type of modeling described earlier in this chapter and will, no doubt, be used more frequently in future studies. 

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