Figure 8: A pentafluorphenyl propyl stationary phase offers greater retention and selectivity for fluoroquinolones than either
C18 or cyano phases, making it well suited for LC–MS analyses.
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Another example of the advantage of a carefully chosen stationary phase for UHPLC is a separation using a pentafluorophenyl
(PFP) propyl stationary phase. This fluorinated phase is extremely retentive and selective for organohalogens or other compounds
containing basic or electronegative functionalities. The analysis of fluoroquinolones such as ciprofloxacin, norfloxacin,
and enrofloxacin demonstrates this characteristic. When compared with C18 and cyanopropyl stationary phases, the PFP propyl
phase is more selective and more retentive (see Figure 8). The US Food and Drug Administration recently announced import controls
on seafood from China, where the use of fluoroquinolones in food animals is permitted. All incoming shipments are now detained
at the border until proven to be free of drug residues. In this specific example, the use of LC–MS–MS is preferred. Using
a PFP propyl stationary phase to heighten the retention of fluoroquinolones is advantageous in LC–MS analysis because it requires
higher organic percentages to elute the compounds. With electrospray ionization (ESI), higher amounts of organic in the mobile
phases help in desolvation, thereby lowering the detection limits. In addition, unwanted adduct formation can be reduced,
and charge competition from matrix interferences are lowered as the fluoroquinolones are better resolved from the matrix components
(3).
Figure 9: Fast, selective analysis of benzodiazepines is made possible by combining the speed of UHPLC with the enhanced selectivity
of a pentafluorophenyl propyl stationary phase.
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In a final example, using the same premise of heightened selectivity of a fluorinated phase toward halogenated drug compounds,
benzodiazepines and two metabolites, a mix commonly assayed on a C18 column, can be resolved in nearly 4 min on a PFP propyl
column (see Figure 9). To get the same level of selectivity from a C18 column, a shallower gradient would be needed, thus
prolonging the analysis time. Because the selectivity of a PFP propyl stationary phase elutes the benzodiazepines in quick
succession, a simple gradient allows for the earlier elution of the more polar metabolites, while maintaining a fast overall
run time.
Conclusion
Although the sub-2-μm particle sizes used in ultrahigh pressure liquid chromatography make fast, highly efficient assays possible,
selectivity is still a critical parameter to consider. The examples reviewed here demonstrate the impact of selectivity on
overall chromatographic resolution and speed of analysis. Proper stationary-phase selection is the most important factor in
optimizing resolution and, with sub-2-μm particles, allows the benefits of UHPLC to be fully realized.
Rick Lake is a pharmaceutical innovations chemist at Restek Corporation, 110 Benner Pike, Bellefonte, PA 16823, tel. 814.353.1300, fax
814.353.1309, rick.lake@restek.com
References
1. R. Freeman, R. Lake, and L. Nolan, "Enhanced Resolution of Endocrine Disrupting Hormones," The Restek Advantage 2006 (3), 8–9, 2006.
2. R. Lake, "Optimize Selectivity and Efficiency in UHPLC Separations," The Restek Advantage 2007 (3), 6–7, 2007.
3. R. Lake, "Simplified LC/MS/MS Analysis of Fluoroquinolones," The Restek Advantage 2007 (1), 6–7, 2007.
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