Determination of the enantiomeric purity of Pemetrexed on the macrocyclic glycopeptides bonded phases

Abstract

The enantioseparation of Pemetrexed on chiral sorbents with macrocyclic glycopeptide antibiotics as chiral selectors was investigated. The pharmacological group of Pemetrexed is antimetabolites with antitumor activity. Pemetrexed is a structural analog of folic acid. IUPAC name of this drug is disodium (2 S )-2-[[4-[2-(2-amino-4-oxo-4,7-dihydro-1 H -pyrrolo[2,3- d ]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioate heptahydrate. The mechanism of Pemetrexed antitumor action is based on the inhibition of the synthesis of purine and pyrimidine. Chiral stationary phases with antibiotics of different structures were used. Columns with silica modified antibiotic eremomycin (commercial column Nautilus-E, Bio-Chem-Mac, Russia) and silica with teicoplanin aglycone (ChirobioticTAG, Astec, USA) were applied. The separation of the enantiomers was carried out by the reversed-phase and polar-ion chromatography modes. The mobile phase in the reversed-phase chromatography mode was a mixture of ammonium dihydrogenphosphate solution and organic solvents (methanol, acetonitrile), and in polar-ion mode – organic solvents (methanol, acetonitrile) with additives of acids (acetic acid, formic acid) and bases (triethylamine, diethylamine). The influence of the components’ nature and concentration in the mobile phase, their composition and pH on the separation of Pemetrexed enantiomers was investigated using the silica modified eremomycin and teicoplanin aglycone. The resolution of Pemetrexed enantiomers was 2.8 - 3.3 in the reverse phase mode on the column with eremomycin. In the polar-ion mode chromatography, Pemetrexed eluted with dead volume (capacity factor k ` = 0.1 ÷ 0.3) independently of the additives concentrations in the mobile phase. The enantiomers of Pemetrexed were not separated on the column with teicoplanin aglycone. Teicoplanin aglycone has one amino-group only, whereas eremomycin has three amino-groups. Electrostatic interactions between teicoplanin aglycone and Pemetrexed were less than interactions between eremomycin and Pemetrexed. Eremomycin has more chiral centers than teicoplanin aglycone. In addition, hydrophobic interactions, hydrogen bonds and dipolar interactions with amide and hydroxyl groups of eremomycin provide high enantioselectivity of sorbent with eremomycin. The determination of enantiomeric purity of Pemetrexed drug on the column with eremomycin was conducted. Mobile phase was (55:15:30) MeOH:ACN:NH 4 H 2 PO 4 (50mM, pH = 2.5). The time of analysis was less than 10 minutes. D-isomer was not detected in the drug. The limit of detection of Pemetrexed D-enantiomer was 0.0003 mg/ml (assumed 3:1 signal to noise ratio) which constituted 0.12 % from the total amount drug. Keywords : macrocyclic glucopeptides, chiral selector, enantiomers, enantiomeric purity of the pharmaceutical substances (Russian) DOI: h ttp://dx.doi.org/10.15826/analitika.2016.20.2.002 E.N. Shapovalova, I.A. Fedorova, A.A. Priporova, I.A. Ananieva, O.A. Shpigun M.V.Lomonosov Moscow State University, 1-3 Leninskie gory, Moscow, , 119991, Russian Federation