###### Abstract  
A simple, cost-effective, and efficient novel high-performance thin-layer chromatography (HPTLC) tool was developed and validated in accordance with International Conference on Harmonisation (ICH) guidelines for the detection and quantification of four fat-soluble vitamins, D\({}_{2}\), D\({}_{3}\), E, and K\({}_{1}\), using chloroform: cyclohexane (55:45, \(\mathit{v}/\mathit{v}\)) as mobile phase. The detection and quantification limits were found to be 30.86 and 93.52 ng/band for vitamin D\({}_{2}\), 19.44 and 58.92 ng/band for vitamin D\({}_{3}\), 14.17 and 42.95 ng/band for vitamin E, and 0.86 and 2.61 ng/band for vitamin K\({}_{1}\), which were similar or lower than those reported in previous methods. The advantage of the developed method is that it uses a simple mobile phase in a single development step and has low detection and quantification limits. The application of the developed HPTLC method was successfully demonstrated with the quantitative analysis of these vitamins in some commercially available pharmaceutical preparations. high-performance thin-layer chromatography; validation; fat-soluble vitamins; quantification; pharmaceutical preparations +
Footnote †: journal: applied sciences  
coupled with diode array /mass spectrometry (LC-DAD-MC) and gas-liquid chromatography (GLC), for the analysis of fat-soluble vitamins in complex matrices like human plasma and serum, pharmaceutical preparations and food items such as fruits and vegetables [2, 6, 7, 8, 9, 10, 11]. In recent years, High-performance thin-layer chromatography (HPTLC) has also been developed into a popular analytical tool based on its simple, multifunctional, highly adaptable and economically efficient analysis process [5]. Moreover, high sample throughput coupled with minimal sample preparation steps facilitate a highly efficient and rapid analysis using HPTLC [4]. In light of the above, HPTLC has become widely popular for the analysis of targeted molecules in various complex matrices like food items (e.g., honey), pharmaceutical formulations, and also nutraceutical preparations [12, 13, 14]. Specifically in the context of fat-soluble vitamins, previous studies found that HPTLC analysis is a \"green\" technique to analyze vitamin D\({}_{3}\) in commercial pharmaceutical products [15]. Another study revealed that using a two-step development coupled with densitometric detection, vitamins A and E as well as some water-soluble vitamins can be determined simultaneously by HPTLC [16] whereas other studies found that fat-soluble vitamins can be effectively analyzed in human plasma and food items using HPTLC [4, 17, 18]. Table 1 provides an overview of previously published HPTLC-based methods for the analysis of fat-soluble vitamins. Reviewing these studies, it can be concluded that there are still some limitations to the quantitative analysis of fat-soluble vitamins either as pure substances or in biological, pharmaceuticals, or food items using existing HPTLC-based methods. For example, they were found to be unable to adequately separate more than two fat-soluble vitamins simultaneously using a single development step. Addressing those limitations, the aim of the current study was to develop and validate a HPTLC-based method for the simultaneous quantification of four fat-soluble vitamins