Separation of nebivolol enantiomers using ultra-high performance convergent chromatography (UPC2)
Saikat Bhattacharya, Dilshad Pullancheri and Gopal Vaidyanathan
Waters India Company (Bangalore, India)
purpose
A fast, simple, and cost-effective method for the separation of nebivolol enantiomers using UltraPerformance Convergence ChromatographyTM ( UPC 2® ) is demonstrated.
background
Nebivolol (Figure 1) is a beta-blocker with nitric oxide-enhancing vasodilatation that can be used to treat high blood pressure. Beta-blockers are the most commonly recommended and prescribed drugs for the treatment of hypertension and heart disease worldwide. Studies have shown that the D-isomer of nebivolol has a major effect on beta selectivity, while the L-isomer may have an antihypertensive effect on the D-isomer. Enantiomeric analysis is usually required to monitor the metabolic pathways of the two isomers. The development of chiral separation methods for such compounds is often time consuming due to the need to screen multiple chiral stationary phases. In general, chiral analytical columns with normal phase solvents have long regeneration times. Solvents used in conventional normal phase chromatography (eg, n-hexane, halogenated solvents, etc.) are toxic, and are expensive to use and dispose of.
Compared to traditional LC methods, the use of UPC 2 to separate nebivolol can save up to 65% of the analytical cost and increase the analytical throughput by up to 80%.
solution
Chiral analysis is usually performed in the early stages of drug development. Convergence chromatography has proven to be a powerful chiral analysis technique due to its superior separation and high analytical speed. The use of convergent chromatography also reduces the use of toxic solvents (usually associated with normal phase LC chiral analysis).
The Waters ® ACQUITY UPC 2 system uses supercritical CO 2 as the primary mobile phase. UPC2 technology fully applies the performance advantages of UPLC ® to supercritical fluid chromatography (SFC). UPC 2 allows screening and development of enantiomer separation methods for pharmaceutical compounds in a very short time while reducing solvent consumption.
This study demonstrates a fast and efficient method for the bulk separation of nebivolol enantiomers using UPC2. In the method presented herein, the nebivolol standard was dissolved in 100% methanol at a concentration of 500 ppm. The ACQUITY UPC2 system is used with a 4.6 x 150 mm, 3 μm chiral column and an ACQUITY UPC 2 PDA detector (wavelength 220 nm).
Using the above procedure, the USP resolution of the nebivolol isomer was 2.1.
The results of multiple consecutive injections showed a high degree of reproducibility, with retention time and peak area %RSD values ​​of 0.1 and 1.0 for the 20 replicate injections, respectively.
to sum up
This experiment demonstrates a simple and efficient method for the separation of nebivolol enantiomers with the ACQUITY UPC 2 system with a 3 μm column. In the experiment, the nebivolol isomer achieved baseline separation within 6 min. The method presented in this paper can save up to 65% of the analysis cost and increase the analysis throughput by up to 80% compared to the traditional LC method. At the same time, the current HPLC positive phase method for nebivolol analysis suggests that the column needs to be equilibrated for 12 to 15 h at the beginning of the analysis, and the column described in this paper only needs to equilibrate for 5 to 10 min in the UPC2 method. . Multiple injection results show that this method is highly reproducible.
The mobile phase used is compatible with the mass spectrometer, thus effectively broadening the range of applications of UPC2 in bioanalytical research.
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