Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin-bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling.
A sensitive and validated liquid chromatography with mass spectrometry method was developed for the enantioseparation of the racemic mixture of pomalidomide, a novel, second-generation immunomodulatory drug, using β-cyclodextrin-bonded stationary phases. Four cyclodextrin columns (β-, hydroxypropyl-β-, carboxymethyl-β-, and sulfobutyl-β-cyclodextrin) were screened and the effects of eluent composition, flow rate, temperature, and organic modifier on enantioseparation were studied. Optimized parameters, offering baseline separation (resolution = 2.70 ± 0.02) were the following: β-cyclodextrin stationary phase, thermostatted at 15°C, and mobile phase consisting of methanol/0.1% acetic acid 10:90 v/v, delivered with 0.8 mL/min flow rate. For the optimized parameter at multiple reaction monitoring mode 274.1-201.0 transition with 20 eV collision energy and 100 V fragmentor voltage the limit of detection and limit of quantitation were 0.75 and 2.00 ng/mL, respectively. Since enantiopure standards were not available, elution order was determined upon comparison of the circular dichroism signals of the separated pomalidomide enantiomers with that of enantiopure thalidomide. The mechanisms underlying the chiral discrimination between the enantiomers were also investigated. Pomalidomide-β-cyclodextrin inclusion complex was characterized using nuclear magnetic resonance spectroscopy and molecular modeling. The thermodynamic aspects of chiral separation were also studied.
Faculty of Pharmacy, University of Medicine and Pharmacy of Tîrgu Mureș, Romania.
Zoltán-István
ZI
Szabó
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary.
Levente
L
Szőcs
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary.
Péter
P
Horváth
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary.
Balázs
B
Komjáti
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary.
József
J
Nagy
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary.
Ádám
Á
Jánoska
Faculty of Pharmacy, University of Medicine and Pharmacy of Tîrgu Mureș, Romania.
Daniela-Lucia
DL
Muntean
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary.
Béla
B
Noszál
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary.
Gergő
G
Tóth
D002851:Chromatography, High Pressure Liquid; D003505:Cyclodextrins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D013237:Stereoisomerism; D013792:Thalidomide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification
D003505:Cyclodextrins; D013792:Thalidomide; C467566:pomalidomide
Chiral separation; Chiral stationary phases; Circular dichroism; Cyclodextrin; NMR spectroscopy