Synthesis and LC-MS Analysis of Cinchona Derivatives
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Cinchona alkaloids with their main component Quinine, are isolated from the barks of several species of cinchona trees and have been used in medicine for over 400 years mainly as anti-malarial medication. These natural alkaloids contain 5 stereo-genic centers and share the same absolute configuration of 3 of these centers. ln 2001, Stork et al published the first stereoselective total synthesis of Quinine, but synthetic Quinine does not cover the global industrial and pharmaceutical demands for Quinine. This project investigated the purity of Quinine tablets that are produced in Norway by Norges Apotekforening (NAF), Kinin NAF® and used as an anti-malarial drug; as well as the purity of 6 raw derivatives of Quinine that are the most abundant Cinchona compounds: Quinine, Quinidine, Cinchonine, Cinchonidine, 10,11-Dihydro- quinine and 10,11-Dihydro-quinidine. Different stationary phases and different combinations of eluents, acidic water, methanol, and acetonitrile were tested. Reversed-phase High Performance Column Chromatography (HPLC) using an Octadecyl stationary phase (C18, ODS) in an acidic mobile phase, and Mass Spectrometry (MS) detection was used. A Difference in retention time behaviors was observed when changing the mobile phase solvents’ combinations and the pH. Results showed that derivatives of Quinidine with an absolute configuration 1S 3R 4S 8R 9S had shorter retention times than their corresponding Quinine derivates with the stereo descriptors of 1S 3R 4S 8S 9R. LC results revealed 8 molecules in a sample that had a mixture of the 6 analytes, indicating the presence of impurities. Different ratios of impurities in the form of dihydro derivatives of Quinine, Quinidine, Cinchonine and Cinchonidine were detected by MS. The analysis of Kinin NAF® revealed that it contains approximately 8% impurities in the form of dihydro and demethoxy derivatives of Quinine. This result was confirmed by Nuclear Magnetic Resonance (NMR) analysis. Synthesis of the two 10,11-dibromo-diastereomers of Quinine was performed to verify the relationship between the molecules’ retention times behavior and their stereochemistry. An LC method was developed to separate the synthesized 2 diastereomeric products using a C18 column with a difference in retention time of more than 2 minutes making it possible to separate them quantitatively so that their absolute configuration on the C10 atom can be determined utilizing NMR and if possible, X-Ray crystallography. Both LC-MS, LC-Time of Flight (LC-TOF) and NMR confirmed a pure form of the mixture of the 2 dibromo diastereomers.