(M0930-09-58) Screening Chiral Stationary Phases in Multiple Separation Modes to Identify a Method for the Enantiomeric Separation of Modafinil

Purpose: Modafinil is an FDA-approved wakefulness-promoting agent that works by increasing the cortical levels of catecholamines; upregulating serotonin, glutamate, orexin, and histamine and decreasing gamma-amino-butyric acid in the brain. Modafinil is a chiral compound composed of R and S enantiomers, which have different half-lives. Enantiopure R-modafinil may be more effective at improving wakefulness due to having a longer pharmacological half-life than racemic Modafinil. Although Modafinil was developed to treat narcolepsy, it is also used off-prescription in dietary supplements for cognitive enhancement (“smart drug”). The purity of these enantiomers offers potential clues about the origin of Modafinil found in adulterated dietary supplements. Indirect Liquid chromatography methods utilizing derivatization and mobile phase additives can be used to successfully resolve enantiomer mixtures. However, both of those solutions can be difficult and cumbersome to implement effectively. Here we show the screening procedure performed using seven different polysaccharide-based chiral columns under reversed phase, normal phase, and polar-organic conditions for a total of 28 chiral screens.

Methods: Columns:
3μ or 5μ Lux Cellulose-1, 100 x 4.6 mm
3μ or 5μ Lux Cellulose-2, 100 x 4.6 mm
3μ or 5μ Lux Cellulose-3, 100 x 4.6 mm
3μ or 5μ Lux i-Amylose-3, 100 x 4.6 mm
3μ or 5μ Lux i-Cellulose-5, 100 x 4.6 mm
3μ or 5μ Lux Amylose-1, 100 x 4.6 mm
5μ Lux i-Amylose-1, 100 x 4.6 mm

Normal Phase Methodology
Mobile Phase:
A: Hexane + 0.1 % Trifluoroacetic Acid
B: Ethanol + 0.1 % Trifluoroacetic Acid
80%: 20% (A:B)

Isocratic Flow Rate: 1.0 mL/min
Column Temp.: Ambient
Instrument: Agilent 1100 Quaternary Pump
Sample: Modafinil standard, 1.0 mg/mL in Acetonitrile

Reverse Phase Methodology
Mobile Phase:
A: Water + 0.1 % Trifluoroacetic Acid
B: Acetonitrile + 0.1 % Trifluoroacetic Acid
40%: 60% (A:B)

Isocratic Flow Rate: 1.0 mL/min
Column Temp.: Ambient
Instrument: Agilent 1100 Quaternary Pump
Sample: Modafinil standard, 1.0 mg/mL in Acetonitrile

Polar Organic 1 Methodology
Mobile Phase:
A: Methanol + 0.1 % Trifluoroacetic Acid
B: Isopropanol + 0.1 % Trifluoroacetic Acid
90%: 10% (A:B)

Isocratic Flow Rate: 1.0 mL/min
Column Temp.: Ambient
Instrument: Agilent 1100 Binary Pump
Sample: Modafinil standard, 1.0 mg/mL in Acetonitrile

Polar Organic 2 Methodology
Mobile Phase:
A: Acetonitrile + 0.1 % Trifluoroacetic Acid
B: Isopropanol + 0.1 % Trifluoroacetic Acid
95%: 5% (A:B)

Isocratic Flow Rate: 1.0 mL/min
Column Temp.: Ambient
Instrument: Agilent 1100 Binary Pump
Sample: Modafinil standard, 1.0 mg/mL in Acetonitrile

Results: Seven CSPs were screened under four different mobile phase systems for a total of 28 screens. It was found that 9 of the 28 screens provided a resolution >1.5. The Lux i-Amylose-3 provided the best separation (Rs = 5.64) under normal phase conditions with an acidic additive. The Lux Cellulose-2 provided the best separation (Rs = 3.93) under reverse phase conditions with an acidic additive. The Lux Cellulose-2 provided the best separation (Rs = 13.58) under polar organic conditions with an acidic additive.

Conclusion: Overall, this study illustrates the powerful utility of a multiple column chiral screening approach coupled with different mobile phase conditions to quickly determine the best column and mobile phase combination to provide an enantiomeric separation. This chiral screening approach has been successfully utilized in our laboratory for a large number of chiral molecules.

Enantiomeric resolution between (R)-modafinil and (S)-modafinil was calculated as follows: