Development of a high-performance thin-layer chromatography method for the rapid quantification of S-equol in biological samples of albino Wistar rats

Equol, an exclusive gut bacteria metabolite, is associated with the incidence of low blood pressure, strokes, and hormone-related cancers due to its estrogen-mimicking structure and antioxidant properties. However, all humans cannot produce S-equol upon consumption of soybean and are thus deprived of the health benefits. Existing methods of equol estimation involve complex instrumentation, which is time-consuming and expensive. Therefore, in this study, we develop a simple, cost-effective, accurate, and rapid high-performance thin-layer chromatography (HPTLC) method for equol estimation. Chromatography was performed on precoated silica gel aluminum 60 F₂₅₄ HPTLC plates, and it produced a compact spot of equol [retardation factor (R_F) value of 0.77 ± 0.04] with a mobile phase ethyl acetate–toluene–formic acid in the ratio 5:4:1 (V/V). Ultraviolet detection was performed densitometrically at λ_max 200 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection, and limit of quantification, following the International Council for Harmonisation (ICH) guidelines. It was found that S-equol concentration was higher in rats fed with fermented soybean, and the concentration was higher in feces as compared with urine samples. The highest S-equol content in feces and urine was 16.74 µg g⁻¹ and 6.34 µg mL⁻¹, respectively. Quantification of S-equol by HPTLC enables us to segregate the human population into two groups: equol producers/healthy individuals and nonequol producers/nonhealthy individuals. In this era of personalized medicine, such an insight will prove to be of great value and also reduce the drug load on our body.

Graphical abstract