Simultaneous determination of retention factors (Rf) of active pharmaceutical ingredients (APIs) with varying polarity using same mobile phase in high performance thin layer chromatography (HPTLC)

The Active Pharmaceutical Ingredient (APIs) selected were Artemether, Lumefantrine, Avobenzone and Ensulizole. The IUPAC names (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-10-methoxy-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin for Artemether, 2-(dibutylamino)-1-[(9Z)-2,7-dichloro-9-[(4-chlorophenyl)methylidene]fluoren-4-yl]ethanol for Lumefantrine, 1-(4-tert-butylphenyl)-3-(4-methoxyphenyl) propane-1,3-dione for Avobenzone and 2-phenylbenzimidazole-5-sulfonic acid for Ensulizole respectively. Artemether and Lumefantrine are used to cure malarial infections caused by parasite Plasmodium falciparum. Avobenzone and Ensulizole are oil-soluble, sun-screen ingredients used to absorb and block harmful UVA rays, thus protecting the skin from sun damage. The APIs vary substantially in polarity in which Artemether is the least polar and Ensulizole being the most polar in nature. HPTLC used for determination was CAMAG Linomat V semi-automatic sample applicator with CAMAG TLC scanner 3 and CATS software for interpretation of data. The stationary phase used was TLC aluminium plates precoated with silica gel 60F 254. The double development approach was employed to improve resolution and peak symmetry among closely eluting APIs. In the first development, methanol was used as the mobile phase followed by a second development using a clear solvent mixture of Toluene: Ethyl acetate: Methanol: Formic acid in a ratio of (6:4:0.1:1). The APIs were dissolved in their respective suitable solvents such that a concentration of 1000 ppm of each API was obtained. A solution of 2.0μl of each API was loaded by using the applicator in which nitrogen was used as a carrier gas that applicators need for this purpose. After the sample application, the plate was kept in Twin Through Chamber (TTC) which was previously saturated with the mobile phase for 20 minutes. The plate was run till the solvent front. It was dried with a dryer before scanning it under the UV lamp. The corresponding spots were observed except for Artemether. The spot corresponding to Artemether was observed after treating it with Anisaldehyde sulphuric acid as a derivatising agent. It was, thus, possible to establish simultaneous determination of Rf values of all the four APIs with a single mobile phase. This will help in the simultaneous quantitative determination of these API formulations and in forced degradation studies and identification of impurities by TLC- Mass spectroscopy.