16
September 2009
ChiralMethod Development Screening Techniques: A practical guide and new approaches in LC-MS
by David S Bell: R&DManager, Supelco/Sigma-Aldrich, Bellefonte, PA.
Dave.Bell@
sial.com DeniseWallworth: European HPLC Specialist, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.
Denise.Wallworth@sial.com
Retention mechanisms in chiral separations are complex and tend to be unpredictable. As a consequence, generic screening techniques utilizing many chiral stationary phases (CSPs) are typically used in method development practices. Normal phase chiral HPLC is commonly used in Drug Discovery and whilst such procedures are well established, there is a strong trend towards more sensitive detection by MS that now calls for a review of mobile phase selection. Additionally, the development of chiral pharmaceuticals has matured, generating larger and larger numbers of chiral molecules in late-stage product development. As a result, chiral LC-MS methods for DMPK and aqueous-compatible/LC-MS friendly methods for pharmaceutical (drug substance) analysis have become even more important.
The key to successful chiral method development is the availability of simple, rapid and reliable screening protocols that avoid the possibility of false positives/negatives. This article outlines a set of simple protocols that uses one of the well-known classes of chiral stationary phases (CSPs), the macrocyclic glycopeptides. CHIROBIOTIC phases are highly compatible with LC-MS and operate in a variety of different mobile phase types.
LC-MS can be used further in method development itself. HPLC combined with tandem MS is often used for the simultaneous quantification of parent drug and metabolites, but it also has the potential for even faster chiral method development. In this article, we also investigate simultaneous chiral method development, injecting a composite set of chiral molecules using LC-MS-MS. By comparing the results from the probe set with that from a single solute, the effect on the screening protocol of simultaneous screening is investigated.
Macrocyclic Glycopeptide phases
Developed in 1994 as a result of research by Dr. Daniel Armstrong1, these innovative CSPs are based on the chemical bonding of macrocyclic glycopeptides on to silica. The glycopeptides used to date include vancomycin, teicoplanin and ristocetin A (commercialised as CHIROBIOTIC™V and V2, T , T2 and TAG, and R, respectively). CHIROBIOTICS CSPs exhibit a large variety of potentially interacting functional groups such as the peptide backbone, amide and chiral sugar moieties for hydrogen bonding and dipole-dipole interactions, plus inclusion sites. Additionally, chloro- substituted aromatic groups provide the
potential for − interactions. However, the unique aspect of these CSPs when compared to other phases is their ionic character arising from carboxylic acid and amine sites providing these phases with exceptional selectivity for ionisable molecules. The individual CSPs available are different from one another in the position, type and number of interactive sites and as
a result offer complementary selectivity. Another result of this variety of interactive sites is that the phases operate in diverse mobile phase conditions, both aqueous and non-aqueous, without the need to reserve one column for each mobile phase. As the mobile phase is changed, the mechanism also changes, providing a wide range of enantiomeric applications for which these phases have become well established. A particular strength in reversed and polar mobile phases results in these phases being especially well-known for their effective interface with MS detection.
Generic screening using glycopeptide chiral phases Because the macrocyclic glycopeptides exhibit enantioselectivity in several mobile phase types, the screening protocol (Table 1) takes advantage of this and includes one run in each type, starting with polar mobile phases followed by reversed phase. Pure EtOH is used as an intermediate solvent in between reversed phase and normal phase and as a polar organic mode. Results from
method development screening of fluoxetine for the first two mobile phases are shown in Figure 1.
The polar ionic mode – a polar organic solvent containing low concentrations of an ionic modifiers - seems unique to these phases and is used for all ionisable molecules. Both acid and base must be added but these can be replaced as part of the optimisation process with MS- and prep HPLC-friendly volatile salts. It is the ratio of acid to base that mainly controls selectivity with bases preferring higher acid and acids, higher base. Reversed phase is used for both ionisable and neutral solutes, while polar organic and normal phase are beneficial for more neutral molecules. The polar ionic mode has many advantages in terms of speed, MS compatibility and sample solubility, but is also beneficial for high throughput prep HPLC. The polar ionic mode is especially useful when solubility in more polar solvents means that normal phase prep is either not possible or inefficient.
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