Class-Selective Enantiomeric Separation of beta-Androgenic Receptors using CHIROBIOTIC™ T Stationary Phase

By: Daniel Shollenberger, Reporter US Volume 26.3

Daniel Shollenberger


The inherent chirality of bio-organic molecules and its affect on biological activity creates a growing need for chiral separations (1). The environmental, food and beverage, and pharmaceutical industries have a need for enantiomerically pure compounds to determine safety, efficacy, and toxicology of products and contaminants. Understanding the role of chirality in pharmacology is extremely important in light of the tragedy of thalidomide use in the 1960s. While one enantiomer of the racemate treated morning sickness, the other caused birth defects and malformity. This event as well as other studies has increased the understanding of chirality in the life sciences. In 1992, the FDA formalized its position on the development of new chiral drugs, now requiring racemate studies to be performed.

Pharmacokinetic, metabolic, and racemic inversion studies are important in the drug development process. Furthermore, with increased pressure for more comprehensive clinical studies, methods that are selective for a class of compounds become attractive. The combination of a class-selective separation with mass spectroscopy provides an informative, high thoughput method with the sensitivity necessary for clinical applications. It also has applications in toxicological screenings.

For this study, we chose β-androgenic receptors as a class of pharmaceutically active compounds. β-androgenic receptors mediate metabolic processes and are important in cardio and pulmonary functions. β-blockers have shown beneficial effects in treating cardiac disorders and tremor, while β-agonists have been used to treat asthma and pulmonary disorders (2). Some β-agonists, like clenbuterol, have been shown to increase muscle mass and decrease adipose tissue. This has made β-agonists popular as a performance enhancer. Moreover, the controversial use in raising livestock has increased concern over contaminated food sources (3).

β-androgenic receptors have in common an alcohol functionality in the beta position relative to a secondary amine (Figure 1). The chiral center of these molecules is located at this site, which is the key interaction for selectivity on a chiral stationary phase. The CHIROBIOTIC T stationary phase was chosen for this study. The CHIROBIOTIC T stationary phase is a teicoplanin macrocyclic glycopeptide bonded silica phase that is capable of ionic, π - π, and hydrogen bonding interactions for the separation of enantiomers. CHIROBIOTIC T has unique selectivity for a number of classes of molecules including carboxylic acids, phenols, neutral aromatic analytes, and cyclic aromatic and aliphatic amines. Moreover, the CHIROBIOTIC T provides chiral separation using reversed-phase solvent mobile phases that are preferred by analytical chemists. Such mobile phases allow for easy transfer to MS (mass spectrometric) as well as ELSD (evaporative light scattering detectors).

Figure 1 Nine β-Receptors Used in this Study

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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 reaction-monitoring (MRM) mode. The nine β-agonists and blockers from Figure 1 were analyzed using the same chromatographic conditions.

Figure 2 Analysis of Four 􀁂-Receptor Compounds using CHIROBIOTIC T in Polar Ionic Mode

Figure 2 shows the UV chromatogram of the separation of four beta-receptor compounds. Clenbuterol elutes first followed by the metoprolol, sotalol, and atenolol. These compounds demonstrate the potential for class selective enantiomeric separation with baseline resolution. The specific conformation of each enantiomer peak was not determined. Table 1 provides chromatographic data including retention times, capacity factor, and selectivity for enantiomer pairs.

Table 1 Peak Descriptions from UV Spectrum of Four Beta Receptor Compounds

Adjustments can be made to flow rate, buffer strength, temperature, and mobile phase composition 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 provide interaction, leading to increased retention and resolution. Changes in buffer strength affect counter ion interaction with the stationary phase. Higher buffer strength decreases retention, while lower buffer strength increases retention. The addition of water to the mobile phase affects polarity of the mobile phase and changes the solvation of 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 show better resolution.

The capability of class selective enantiomeric separation was further explored by utilizing mass spectrometric detection. LC-MS provides enhanced sensitivity as well as MS selectivity. Table 2 provides a summary of results from the analysis of nine β-receptor compounds by mass spectrometry. The extracted ion 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 β-Receptor Compounds by LC-MS on CHIROBIOTIC T

The column used in both analyses is a 25 cm x 4.6 mm I.D. at a flow 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 optimized for this higher flow 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 flow rate. While a direct transfer may not always be possible, the use of UV detection for chromatographic method development is useful before optimization of LC-MS conditions for trace analysis.

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This application demonstrates a chiral separation in a mobile phase system preferred with LC-MS analysis. The separation of compounds with similar functionality and pharmacological properties by one method has many benefits for clinical and toxilogical applications. It increases sample throughput and has increased sensitivity with MS detection. Future experiments with column dimension may lead to faster LC-MS separations for chiral analytes.

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  1. Chiral Liquid Chromatography; Lough, W.J., Eds.; Chapmen&Hall: Glascow, UK, 1995; pp 15-35.
  2. Lee, H.B.; Sarafin, K.; Peart, T.E.; J. Chromatog A.; 2007, 1148, 158-167.
  3. Blomgren, A.; Berggren, C.; Holmber, A.; Larsson, F.; Sellergren, B.; Ensing, K.; J. Chromatog. A.; 2002, 975, 157-164.

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