Comparison of the limits of detection and quantification of estradiol hemihydrate determined by thin-layer chromatography using different chromatographic conditions

ABSTRACT

Thin-layer chromatography (TLC) combined with densitometry was used to determine the limit of detection (LOD) and quantification (LOQ) of estradiol hemihydrate. Average values of both LOD and also LOQ were determined on the basis of the standard deviation of the response and slope of special calibration curve in accordance with the procedure required by validation guidelines. Influence of chromatographic conditions, such as different sorbents and various mobile-phase compositions on determining of LODs and LOQs of examined steroid by normal phase TLC (NP-TLC) and reversed-phase (RP-TLC) techniques, was estimated. Of all chromatographic plates and mobile phases used, those that gave the best results (the lowest LOD and LOQ value) using NP-TLC system are silica gel 60 F₂₅₄ plates developed with mobile phase consisted of dichloromethane–ethyl acetate (8:2, v/v). In the case of RP-TLC method, a mixture of methanol–water (9:1, v/v) as mobile phase enabled detection of estradiol hemihydrate at the lowest concentration level on RP8 F_254s precoated plates. The results of LODs and LOQs obtained in this study confirm that the proposed TLC-densitometric method is suitable for the quantification of estradiol hemihydrate in the samples containing this steroid in the range of 0.093–3.930 µg spot⁻¹. The described method could be an important tool in pharmaceutical as well as in clinical or environmental analysis of examined steroid hormone.

KEYWORDS:

• Estradiol hemihydrate
• limit of detection
• limit of quantification
• steroid hormones
• TLC densitometry