Chiral method development

Chiral method development is often referred to as one of the most difficult fields in terms of development time. Interaction with a chiral selector is required to achieve separation but the enantioselectivity of a given selector for a given chiral molecule is a priori unknown. For some compounds, it can take several days to find suitable separation conditions when using sequential approaches. Therefore, industry most often defines generic separation strategies, which are often kept internally or are... 

In this way, we aim to give an overview of what can be used as a separation technique and which conditions will most likely give an (beginning of) enantiomer separation after a first screening. Chiral method development starter kits are also available and evaluated in some papers [2], but we will not focus on this kind of applications. 

Another application of rapid chiral method development in SFC was presented by Villeneuve and Anderegg [50]. The same columns as in Reference 49 are used, combined with four organic modifiers, including methanol, methanol with 0.1% TEA, ethanol, and isopropanol. Using a six-way column switcher, the four columns can remain constantly inside the device. For some separations, 0.1% DEA or TEA was added. The separations were generated at a flow rate of 2 mL/min, 205 atm of pressure, and a temperature of 40°C. This approach seems applicable, based on the baseline separation that was obtained for the four analyzed compounds, but lacks detailed sequential steps. Therefore, no real strategy can be derived. 

Mangelings, D., Maftouh, M., Massart, D.L., Vander Heyden, Y. Generic capillary electrochromatographic screening and optimization strategies for chiral method development. LC-GC Europe, 2006, 19, 40 7. 

P/ACE System MDQ chiral methods development P/ACE System MDQ highly sulfated cyclodextrin trial kit eCAP Amine Capillary Method Development Kit/Small Molecules eCAP Carbohydrate Labeling Kit... 

Sajonz, R, Gong, X., Leonard, W. R., Jr., Biba, M., and Welch, C. 1., Multiparallel chiral method development screening using an 8-chaimel microfluidic HRLC system. Chirality 18(10), 803-813, 2006. 

Many researchers have put a considerable amount of effort into studies of the chiral recognition mechanisms (using, e.g., NMR and molecular modeling), but yet the choice of chiral selector or chiral phase for a new compound is often based on trial and error. Different strategies for chiral method development have been presented by many of the retailers of chiral columns as a service for the customers. In addition to the information supplied by these retailers, another source of knowledge is Chirbase, a database that contains more than 50,000 HPLC separations of more than 15,000 different chiral substances [61], which also can provide guidance to the analytical chemist. 

Application databases have been particularly popular in the world of chiral method development (Figure 10-5). While it has been observed that small changes in compounds can result in loss of effectiveness (separation selectivity) for a given method, the results of searches can be used to create targeted method screens that can reduce the time and expense of development [36]. 

Table 1 Initial Conditions for Chiral Method Development Using Modified Carbon Dioxide as the Mobile Phase...

The column oven tends to be a cross between a GC and an HPLC oven. It is now common to mount a column selection valve in the oven with 6 or even 12 columns, particularly when performing chiral method development. 

Fully Automated Chiral Method Development Stations for HPLC and SEC (includes a Berger SFC and Chiral Advanced Laser Polarimeter), Gary W. Yanik, PDR-Chiral. 

Supported liquid membranes (SLMs) consisting of 5% tri-n-octylphosphine oxide (TOPO) dissolved in di-w-hexylether/n-undecane (1 1) have been used in the simultaneous extraction of a mixture of three stUbene compounds (dienestrol, diethylstilbestrol, and hexestrol) in cow s milk, urine, bovine kidney, and liver tissue matrices [183]. The efficiencies obtained after the enrichment of 1 ng/1 stilbenes in a variety of biological matrices of milk, urine, liver, kidney, and water were 60-70, 71-86, 69-80, 63-74, and 72-93%, respectively. A new method to contribute to the discrimination of polyphenols including resveratrol with synthetic pores was proposed [184]. The work [185] evaluated two types of commonly available chiral detectors for their possible use in chiral method development and screening polarimeters and CD detectors. Linearity, precision, and the limit of detection (LOD) of six compounds (trans-stilbene oxide, ethyl chrysanthemate, propranolol, 1-methyl-2-tetralone, naproxen, and methyl methionine) on four common detectors (three polarimeters and one CD detector) were experimentally determined and the limit of quantitation calculated from the experimental LOD. trans-Stilbene oxide worked well across all the detectors, showing good linearity, precision, and low detection limits. However, the other five compounds proved to be more discriminating and showed that the CD detector performed better as a detector for chiral screens than the polarimeters. 

Linearity, precision, and the limit of detection (LOD) of trans-stilbene oxide and other compounds were investigated [89]. The authors investigated the second factor and evaluated two types of commonly available chiral detectors for their possible use in chiral method development and screening polarimeters and CD detectors. It was shown that frans-stilbene oxide worked well across all the detectors examined, showing good linearity, precision, and low detection limits. 

Table 1 Initial conditions for chiral method development using modified carhon dioxide as the mobile phase.

Unlike achiral column chromatography, chiral chromatography does not follow a direct and predictive Snyder and Kirkland method development strategy. This is due to the need for chiral method development to first identify stationary... 

The basis of chiral method development screening strategies is largely dictated by whether the method is to be applied to analytical or preparative applications. Preparative chromatography tends to use volatile modifiers and mobile phases for easy removal after sample collection. Sample isolation capabilities have led to the advancement of chiral SFC since CO2 is easily driven off. Chiral separations in preparative chromatography only need to be optimized to a point where pure samples can be collected. Conversely, while analytical chromatography can use a greater variety of modifiers and mobile phases, the separation needs to be optimized to a point where suitable resolution (R > 1.5) is achieved between the enantiomers of interest. 

TABLE 7 Commonly Used Modifiers in Chromatographic Chiral Method Development... 

Reversed phase chiral separations are desired simply for efficiency in generating results from laboratories whose instrumentation is routinely configured to run in reversed and not normal phase modes.Normal phase conditions are less attractive to the analytical chemist for this reason and deter laboratory efficiency. Typical commercial chiral LC columns found on pharmaceutical reversed phase LC chiral method development screens are listed in Table 8. Table 11 shows suggested chromatographic conditions employed in reversed phase chiral screening. 

Figure 3 illustrates an orthogonal approach to chiral method development. This tactic resulted from the combined efforts and experience supplied by many pharmaceutical vendor and development laboratories. Each of the sections can... 

FIGURE 3 Unified chiral screening approach to chiral methods development. 

Regardless of whether a traditional normal phase LC, reversed phased LC, or SFC format is used, routine analytical chiral method development screening is very effective and efficient for developing chiral selective methods in the pharmaceutical industry. 

Wong, M. M., Holzheuer, W. B. and Webster, G. K. A Comparison of HPLC and SFC Chiral Method Development Screening Approaches for Compounds of Pharmaceutical Interest. Curr. Pharm. Anal 4 101, 2008. 

A knowledge-based chiral method development strategy can be very effective on chiral column selection. For example, a chiral screening method was used to monitor the enantiomeric purity of an atropisomeric dmg candidate. It returned the Chiralcel OD-RH column in reversed phase with a baseline enantioseparation of both atropi-somers (Fig. 12a). There was a need to redevelop the chiral method as the compound moved into development stages. Based on possible interaction sites around the chiral asymmetric axis of the compound (a primary amine for ionic and H-bonding interactions and an aromatic ring for k-k interaction), Chirobiotic V2 was tested since... 

To illustrate the complexity of chiral method development, the case of the anticancer brivanib alaninate and its isomers is discussed [150,166]. Several structural features of brivanib make its enantiomeric separation extremely difficult to achieve. First,... 

There are many classes of CSPs applicable in different mobile-phase modes. In particular, CSPs based on derivatized polysaccharides, native and derivatized cyclodextrins, macrocyclic glycopeptides, and Pirkle-type chiral selectors operate quite well in four separation modes, i.e RP, polar organic phase, NP, and super- or subcritical fluid chromatography (SFC) conditions. It is common that a chiral compound can be separated on the same CSP in more than one separation mode [58, 160, 166, 170-176]. For example, Nutlin-3, a small molecule antagonist of MDM2, has been baseline resolved from its enantiomer in all four mobile-phase conditions (Fig. 16) [170]. Multimodal enantioseparation on the same CSP would be greatly beneflcial for chiral method development in pharmaceutical industry. 

AMn A, Antosz FJ, Ausec JL, Greve KF, Johnson RL, Mag-nusson L-E, Ramstad T, Secreast SL, Seihert DS, Webster GK. An orthogonal approach to chiral method development screening. Curr. Pharm. Anal. 2007 3 53-70. 

Sajonz P, Gong X, Leonard Jr.WR, Biha M, Welch CJ. Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system. Chirality 2006 18 803-813. 

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