Understanding the role of chirality in pharmacology became extremely important following the tragedy of thalidomide use in the 1960s. At that time the effect of chirality was poorly understood, and whilst it was discovered that the sedative effect resided in the R- enantiomer and the teratogenic effect in the S-, it is now known that there was some evidence of a mutual synergistic effect between the two enantiomers for both the effects of this drug, along with additional issues of neurotoxicity. This complex situation has fortunately resulted in the development of effective chiral chromatog raphy solutions.
Furthermore, with increased pressure from rising numbers in chiral drug candidates (over 80 % of new chemical entities are now single enantiomers (1)), columns that are selective for a class of compounds become attractive and more useful: when combined with mass spectroscopy, informative, high throughput methods with the necessary sensitivity for clinical toxicological applications are obtained.
For this study, we chose b-blockers and b-agonists as a class of pharmaceutically active compounds. b-androgenic receptors mediate metabolic processes and are important in cardio and pulmonary functions. b-blockers have shown benefi cial effects in treating cardiac disorders and tremor, while b-agonists have been used to treat asthma and pulmonary disorders (2). Some b-agonists, like clenbuterol, have been shown to increase muscle mass and decrease adipose tissue. This has made b-agonists popular as a performance enhancer. Moreover, the controversial use in raising livestock has increased concern over contaminated food sources (3).
b-blockers and b-agonists have in common a chiral alcohol functionality in the beta position relative to a secondary amine (Figure 1). The CHIROBIOTIC T chiral stationary phase (CSP) was chosen for this study following many recent studies using this CSP(4). This silica phase is bonded with teicoplanin macrocyclic glycopeptide that is capable of ionic, p-p, and hydrogen bonding interactions, making it broadly applicable to a wide range of molecular classes. Examples of observed unique selectivity include carboxylic acids, phenols, neutral aromatic analytes, amino acids and cyclic aromatic and aliphatic amines. Moreover, the CHIROBIOTIC T provides the majority of its chiral separations using reversed-phase or polar mobile phases that are preferred by many bioanalytical, stability and formulation chemists. Such mobile phases also allow for easy transfer to mass spectrometric detection (MS), as well as evaporative light scattering detectors (ELSD).
Figure 1.Nine b-Blockers and b-Agonists Used in this Study.
This application was developed on a Hitachi® L-2300 chromatograph with UV detection using a CHIROBIOTIC T, 25 cm x 4.6 mm I.D. column, 5 μm silica. The mobile phase was 15 mM ammonium formate in methanol with a column temperature of 25 °C. Atenolol, clenbuterol, metoprolol, and sotalol at a concentration of 1.0 mg/mL were separated under these conditions (Figure 2). The method was then transferred to an Agilent® 1100 equipped with an Applied Biosystems 3200 Q-Trap mass spectrometer run in multiple reactionmonitoring (MRM) mode. The nine b-agonists and blockers from Figure 1 were analysed using the same chromatographic conditions.
Figure 2.Analysis of Four b-Receptor Compounds using CHIROBIOTIC T in the Polar Ionic Mode.
Figure 2 shows the UV chromatogram of the separation of four b-blocker and b-agonist compounds in a single run. Clenbuterol elutes fi rst followed by the metoprolol, sotalol, and atenolol. These compounds demonstrate the capability of these CSPs for class select ive enantiomeric separations with baseline resolution. The specifi c conformation of each enantiomer peak was not determined. Table 1 provides chromatographic data including retention times, capacity factor, and selectivity for enantiomer pairs.
The mobile phase used for this separation is a simple 100 % methanol with added volatile salt (the polar ionic mode). Adjustments can be made to fl ow rate, salt concentration and temperature based on the requirements of the analysis. Typically in this mode, decreased flow rate enhances residence time between the analyte and stationary phase thereby increasing resolution. A decrease in temperature seems to increase interaction, leading to increased retention and resolution. Changes in salt concentration affect the counter ion interaction with the ionic sites of the stationary phase. Higher salt concentration decreases retention, while a lower one increases retention. The addition of water to the mobile phase in this polar ionic mode (up to 10 %) affects the polarity of the mobile phase and changes the solvation of both analytes and stationary phase. In some cases, this has shown a decrease in retention, but an increase in enantiomeric resolution. Decreased injection volume and loading often also provide increased resolution.
The capability of class selective enantiomeric separation was further explored by utilising mass spectrometric detection. LC-MS provides enhanced sensitivity as well as MS selectivity. Table 2 provides a summary of results from the analysis of eight receptor compounds by mass spectrometry. The extractedion chromatograms for each mass transition are shown in Table 2. The retention times for each peak, the capacity factor, and selectivity between enantiomer peaks are also given. All of the compounds have been identified and each enantiomeric pair separated with good selectivity. The chromatographic conditions are identical to those in Figure 2, with only the sample concentrations and injection volumes varying based on the detector.
Table 2. Individual Extracted Ion Chromatograms from Analysis of Nine b-Receptor Compounds by LC-MS on CHIROBIOTIC T
The column used in both UV and MS analyses is a 25 cm x 4.6 mm I.D. at a fl ow of 1 mL/min. This is a much larger column than is typically used in an LC-MS analysis. Source gas and capillary adjustments were optimised for this higher fl ow rate. This method shows the direct transfer of chromatographic conditions between detection techniques, but the LC-MS analysis could easily have been transferred to a smaller column length and internal diameter at a lower fl ow rate. While a direct transfer may not always be possible, the use of UV detection for chromatographic method development is useful before optimisation of LC-MS conditions for trace analysis.