Comparison the separation of four selected immunosuppressants in different thin-layer chromatographic conditions

Introduction Thin-layer chromatography (TLC) is a useful tool that enables the separation of the components of a mixture for qualitative and quantitative analysis. The aim of this study was to find the conditions for separation of four immunosuppressants, such as everolimus, zotarolimus, tacrolimus and temsirolimus.

Methods TLC was performed on 10 x10 cm plates precoated with silica gel 60F₂₅₄, silica gel 60 with kieselguhr 254 and silica gel RP-18F₂₅₄. Five microliters of ethanol solution (1 µg/µL) of each drug was applied to the plate using micropipettes. The chromatograms were developed by using mobile phases composed of toluene/acetonitrile/acetic acid (6:4:0.1 v/v/v; F1), chloroform/toluene/methanol/glacial acetic acid (4:4:2:0.1 v/v/v/v; F2), propan-2-ol/water (4:1 v/v; F3), acetonitrile/methanol/propan-2-ol (4:3:3 v/v/v; F4), ethanol/water (4:1 v/v; F5), acetonitrile/water (4:1 v/v; F6) and methanol/water/formic acid (36:14:0.1 v/v/v; F7). Chromatograms showing spots with a diameter of 5-7 mm were visualized at 254 nm.

Results TLC analyses showed the influence of the applied mobile and stationary phases on the values of the retardation factor (R_f) and separation parameters of the pairs of examined compounds. The highest R_f values (close to 0.99) for studied compounds, not allowed for their separation were obtained with phase F7 and RP18F254 plates. The optimization of separation conditions indicates that the best separation was achieved with the mobile phase F4 and RP18F₂₅₄ plates. Under these conditions, the highest separation factors for the three studied pairs, namely ΔR_f: 0.06-0.25; R_S: 1.08-1.36, a: 1.37-8.14 and R_f^α: 1.08-3.00, were obtained.

Conclusions The optimized TLC conditions consisting of mobile phase F4 and RP18F₂₅₄ plates may be applicable to the quality control of drugs under study. The developed method is fast and economic. It allows for the analysis of four tested compounds simultaneously.