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MS coupling enhances chiral method development: smaller particles and the addition of ms detection help speed chiral analysis time and accelerate method development.

Chirality plays a critical role in drug efficacy because enantiomers may have vastly different biological, pharmacological and toxicological properties. The majority of newly introduced drugs are chiral, and, by 2020, according to a report by Global Industry Analysts, Inc., it is expected that nearly 95% of pharmaceutical drugs will be chiral. To keep pace with this growth, researchers in drug discovery are seeking faster methods to enable higher throughput enantioselective analyses, enantiomeric excess determination and impurity profiling of chiral molecules.

Separation of enantiomers (compounds with the same molecular weight that are mirror images of each other) poses a difficult chromatographic challenge. Chiral separation method development is typically less predictable than achiral method development. Small differences in molecular structure can significantly change resolution for a given column/mobile phase system. Furthermore, analysts often encounter many structurally diverse stereoisomers with purity levels varying from 70 to 90 percent.

Chiral method development is traditionally accomplished by tediously testing various column chemistry and mobile phase combinations, using normal-phase liquid chromatography (NPLC) to find a system that will separate the enantiomers of interest. Then, the mobile phase and gradient composition is fine-tuned to optimize the method. Supercritical-fluid chromatography (SFC), a form of NPLC, has shown superior resolving power in chiral separations and obviates the use of toxic solvents, such as hexane and chloroform, typically associated with NPLC. SFC mobile phases are primarily compressed carbon dioxide to which may be added small amounts of a co-solvent, such as methanol. However, the inability to meter supercritical C[O.sub.2] and control key parameters such as temperature, pressure, flow rate and gradient composition--reliably and reproducibly--complicates and slows SFC method development. This has hindered the adoption of SFC as a routine analytical tool.

Waters' UltraPerformance Convergence Chromatography ([UPC.sup.2]) system raises the performance bar for NPLC separations using dense gas C[O.sub.2] as the mobile phase. It has the ability--especially by using small-particle columns and coupling the high specificity of mass spectrometry with UV detection--to shorten separation cycle time, thereby facilitating and speeding up chiral separation methods development. The [UPC.sup.2] system combines the best features of LC and GC: higher selectivity via orthogonal modes of separation and higher mobile phase diffusion and efficiency, respectively.

When adequately compressed and made dense, C[O.sub.2] becomes a green, non-toxic, inexpensive alternative to HPLC-grade solvents, especially those toxic organic liquids that incur significant disposal costs. As the primary [UPC.sup.2] mobile phase, its low viscosity may decrease operating pressure while increasing efficiency for a given particle size and linear velocity. Compared to LC solvents, mass transfer in supercritical-fluid C[O.sup.2] is enhanced. Its critical temperature (31 C) is far lower than typical GC operating temperatures, so, as with LC, heat-labile compounds may be analyzed. Perhaps the most significant advantage of C[O.sup.2] may be economy. It reduces the cost of the mobile phase used per sample analysis from dollars to pennies.

The [UPC.sup.2] system meters highly compressed C[O.sup.2] reproducibly and reliably, and regulates pressure, temperature and mobile phase gradient composition. This is key to maintaining fluid density and stabilizing analyte retention on sub-two micron particle columns. These parameters may be used in conjunction with a broad polarity spectrum of co-solvents and available column chemistries to fine-tune resolution and selectivity for successful separations. Furthermore, low system dispersion enables an increase in speed, sensitivity and resolution inherent in small-particle columns.

Smaller particles

Flurbiprofen is an anti-inflammatory used in pain management. It has one chiral site, as indicated in the structures in Figure 1. Using an [UPC.sup.2] System with photodiode array (PDA) detection, a simple mobile phase (C[O.sup.2]: methanol 75:25) at a back pressure set point of 130 bar, and a column temperature of 40 C, an excellent separation of flurbiprofen enantiomers is obtained. As shown in Figure 1, smaller particle size not only speeds the analysis but also reduces solvent consumption.

Clenbuterol is a potent bronchodilator used to treat people with chronic breathing disorders, such as asthma. Typical daily dosage is 2 to 40 mg/day, so sensitive methods of analysis are needed to determine its enantiomers at low concentration. Using a [UPC.sup.2] system, a matrix of 16 gradient separations using combinations of four different columns and four co-solvents was run in less than three hours. Within a few more minutes, the best combination was used to obtain the optimized isocratic separation shown in Figure 2.

Retention time and peak area reproducibility were excellent (% RSDs [n=6] of 0.012 and 0.016 for retention time, 0.34 and 0.33 for area, of peaks 1 and 2, respectively). If more specificity and sensitivity is desired for bioanalysis, the volatile mobile phase is compatible with MS detection.

MS coupling

Unlike NPLC, [UPC.sup.2] separations use MS-compatible co-solvents, such as alcohols, thereby enabling the application of highly specific MS detection to chiral analysis. By tuning in to a selected mass characteristic of a known chiral analyte, MS detection can locate, within a mixture of compounds, its pair of enantiomers, though it cannot distinguish which of the pair each peak represents. Such specificity may confirm if the enantiomers co-elute with a related compound or process impurity. Further, the high sensitivity of MS detection may detect very low levels of one enantiomer in the presence of the other. This capability can be used to ensure that enantiomeric excess falls within acceptable levels.

When sample mixtures are complicated by the presence of several enantiomeric pairs that must be determined, there are two options for increasing analytical throughput. With a significant capital investment, a sample containing several chiral compounds may be screened simultaneously on multiple systems, each of which uses a column/mobile phase combination specific for one of the analytes. A more sophisticated and less costly approach uses a single [UPC.sup.2]-MS combination to screen a sample for multiple analytes in a single analysis, identifying the enantiomeric pairs by examining the extracted ion chromatograms. This idea is illustrated in principle by the model experiment in Figure 3.

Waters Corp., Milford, Mass.
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Publication:Chromatography Techniques
Geographic Code:1USA
Date:Jun 1, 2013
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