The Development and Validation of HPLC and HPTLC-Densitometry Methods for the Determination of 1, 4-Naphthoquinone Content in Eleutherine Bulbosa Extract

Abstract

Objective: The aim of this research was to develop and validate identification and Quantitation methods for 1,4-naphthoquinone in the extract of Eleutherine bulbosa. Methods: High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography with densitometric detection (HPTLC-densitometry) were employed as analytical techniques. HPTLC-densitometry was performed at a wavelength of 249 nm, while UHPLC was conducted at a wavelength of 254 nm. Both methods were utilized to analyze 1,4-naphthoquinone in 96% ethanol extract of E. bulbosa as a Quantitation parameter in the standardization of the formulation. HPTLC separation was carried out on a 20 cm × 20 cm HPTLC glass plate coated with silica gel 60 F254 using a mobile phase of chloroform: methanol (8:2, v/v). For HPLC analysis, a C18 column with an isocratic method was employed using a mobile phase of 95% methanol in pump A and 0.5% chloroform in pump B. The calibration curve of peak area against concentration showed linearity within the range of 2500-15000 ppm/spot−1 and 3–21 μg/ml for HPTLC and HPLC, respectively. Both methods were validated for accuracy, precision, linearity, selectivity, LOD, and LOQ. Results: The results showed that both methods exhibited linearity that met the standards, as they produced correlation coefficients (r) greater than 0.9900. Furthermore, both methods demonstrated good accuracy, with consecutive recovery values of 99.53% and 101.89%. On the other hand, the methods fulfilled the precision requirements, with respective values of 0.7159% and 2.884% (in compliance with the requirement of<5%). Additionally, to meet the LOD and LOQ requirements in HPTLC, the LOD value obtained was 163 ppm, and the LOQ value was 495 ppm. In HPLC, the retention time of the standard 1,4-naphthoquinone and the analyte compound in the extract of E. bulbosa were the same, at 3.507 min. The selectivity test on HPTLC indicated that the 1,4-naphthoquinone compound was at an RF value of 0.81, which was also detected in the extract of E. bulbosa at the same RF value. Conclusion: In conclusion, our findings demonstrate that HPLC and HPTLC methods for the determination of 1,4-naphthoquinone content have met the standards for linearity, accuracy, precision, selectivity, LOD, and LOQ. Therefore, these methods can be recommended for the quality control of raw materials of E. bulbosa extract.