Reversed-phase Preparative Liquid Chromatography Research Articles

Gram-Scale Purification of Two Sesquiterpene Lactones from Chartolepsis Intermedia Boiss.

Two sesquiterpene lactones, grosheimin and cynaropicrin, present in the aerial part of the Chartolepis intermedia Boiss. plant can be obtained from the ethyl acetate solid extract of the dried plant. A large-scale purification processing 16 kg of dry plant is presented. The first liquid–liquid extraction step of the 16 kg produced 550 g of dry extract that were divided in two equal portions. A classical low-pressure chromatography method can produce from 275 g of dry extract 13.8 g of grosheimin and 52.3 g of cynaropicrin 95 % pure lactones in 110 h using large amounts of organic solvents and 8 kg of silica gel. Another purification method employing countercurrent chromatography, with a modern hydrostatic column, and reversed phase preparative liquid chromatography were used with the second 275 g dry extract portion. It could produce 17.9 g of 99.4 % pure grosheimin and 68 g of 98.7 % pure cynaropicrin lactones in 95 h, with better yields and minimized losses in chromatographic columns, and using smaller solvent volumes than the classical method. The two purification processes are fully described and compared.

HPLC separation and in vitro antimalarial studies of Artemisia annua plants from two different origins: Cameroon versus Luxembourg.

Malaria is a devastating disease, particularly in Africa, due to development of resistance by Plasmodium falciparum against all known antimalarial drugs, including artemisinin. Therefore, the search for new antimalarial drugs is urgently needed, especially drugs that can impede the heme detoxification pathway in the malaria parasite, a crucial requirement for parasite survival in host erythrocytes. Water infusions of Artemisia annua plants from two different origins, Cameroon and Luxembourg, were used in this study. A semi-quantitative in vitro method, based on the inhibition of ferriprotoporphyrin IX (FP) biomineralisation developed by Deharo et al. [16], was used to reveal the differences in antimalarial activity of both plants. Reversed phase preparative liquid chromatography coupled to a photo diode array (PDA) detector was also used to test for differences in antimalarial activity. Water extracts from the leaves of the Cameroon plant showed a higher potential antimalarial activity, represented by a higher ability to inhibit β-haematin formation in vitro than A. annua extracts from Luxembourg. Although extracts of the plants of both origins showed comparable efficiencies at high concentrations, the absorbance value at 405 nm of a 10% dilution of the Cameroon plant extract was 0.075, whereas it was 1.515 for the Luxembourg plant extract. The absorbance is inversely proportional to the antimalarial activity. According to the Prep-HPLC chromatogram of the Cameroon crude sample, seven major compounds at 325 nm were found. However, only four much less pronounced compounds appeared in the Luxembourg crude sample under the same chromatographic conditions and concentration. These were preliminarily identified as polyphenolic compounds. A. annua infusions are widely used by people who cannot afford other treatments. Depending on the cultivation locality different chemical profiles exist. This results in differences in hemozoin formation and will therefore also lead to alterations in antimalarial activity.

Method development of reversed-phase preparative liquid chromatography for insulin

The development and optimization of reversed-phase preparative liquid chromatography method for insulin were performed. The chromatographic retention, sample loading and peak broadening were investigated. The mobile phase gradient conditions and stationary phases were optimized. The parameters of the method contained the peak broadening levels under different amounts of sample loading and the concentration distribution of the target compound in the elution curves. The parameters of peak broadening levels were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The most suitable chromatographic system (including stationary phases and mobile phase conditions) was selected. A gradient program with slow change of the strong elution solvent was used. The most suitable stationary phase should exhibit the narrower peak broadening and the peaks were best to broaden to both sides comparing to that under the analytical conditions. Besides, the concentration distribution of the target compound should be focused on the middle of the elution. The guide principles were validation by purification of crude insulin products. The preparative chromatographic system constructed through this method can remove impurities effectively. The method has good practical value. It can provide reference for the development of preparative chromatographic methods of other macromolecular compounds.

Potential Antimalarial Activity from Alcoholic Extracts of Wild <em>Salvia palaestina</em> Leaves

Malaria threatens the lives of more than one third of the world’s population; it is a major cause of human deaths. As a result of the emergence of resistant strains of <em>Plasmodium falciparum</em> to common antimalarial drugs, the search for new antimalarial drugs is urgently needed. Hemozoin synthesis is an indispensable process for the parasite survival and is the target of action for several known antimalarial drugs. Sage, <em>Salvia palaestina</em>, is an aromatic Mediterranean plant. Its leaves have been used over centuries in Palestinian traditional medicine and are now being investigated for potential antimalarial activity. This study reveals the antimalarial activity of crude and HPLC separated fractions tested using two methods; the inhibition of ferriprotoporphyrin IX (FP) bio- mineralization: semi-quantitative micro-assay used by Deharo and a previously self-developed quantitative in vitro method. Reversed phase preparative liquid chromatography coupled to Photo Diode Array (PDA) detector was used to isolate and enrich eight fractions. Three fractions showed promising antimalarial activity. The crude alcoholic extract of sage leaves seems to have the potential of an antimalarial drug; it prevents β-hematin formation with an efficiency of about 72% when compared to the standard Chloroquine which gave 93% at comparable concentrations of chloroquine and extract.

Isolation and structural elucidation of two impurities from a diacerein bulk drug

Two impurities were found in the crude sample of diacerein. The level of these impurities 1.14% and 1.24% were detected by isocratic reverse-phase high performance liquid chromatography (HPLC). The molecular weights of the impurities were determined by liquid chromatography–mass spectroscopy (LC–MS) analysis. These impurities were isolated from crude sample of diacerein by reverse-phase preparative liquid chromatography. These impurities were characterized as 5-acetoxy-4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid (Impurity-1) and 4-acetoxy-5-hydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid (Impurity-2) respectively. Structural elucidation of both the impurities were carried out by 1H NMR, 13C NMR, DEPT, 1D NOESY, MS and IR spectroscopy.

Identification, isolation and characterization of potential degradation product in risperidone tablets

One unknown impurity (degradation product) present at a level below 0.1% in the initial samples increased to a level of 0.5% in 6 M/40 °C/75% RH stability samples of risperidone tablets was detected by gradient reverse-phase high-performance liquid chromatography (HPLC). This impurity was isolated using reverse-phase preparative liquid chromatography. Based on the spectral data the structure of this impurity is characterized as 3-[2-[4-[6-fluoro-1,3-benzoxazol-2-yl]piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro-4 H-pyrido[1,2- a] pyrimidin-4-one. Structural elucidation of this impurity by spectral data ( 1H NMR, 13C NMR, DEPT, MS and IR), formation and mechanism has been discussed in detail.

A Low Cost Gram Quantity Preparative Reverse Phase Chromatography System

Abstract The preparation of a low cost reverse phase preparative liquid chromatography system is described. The described system is capable of providing gram quantity scale-up from analytical reverse phase hplc systems. Load capacity is in the two gram range.