Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

Abstract

Objective:The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga.Materials and Methods:RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase.Results:The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin.Conclusions:The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient.

A new multiresidue thin layer chromatography–ultraviolet detection method for the rapid determination of six herbicides residue applied as ready mix formulation for rice

“…TLC method validation was performed following the guidelines set by the European Commission, [32], the International Conference on Harmonization , [33], the International Union of Pure and Applied Chemistry [34], and EURACHEM guidelines, [35] and as followed by Kamal et al. [36] and results are presented in Table 2. A standard solution of pyrazosulfuron‐ethyl, pretilachlor, cyhalofop‐butyl, penoxsulam, ethoxysulfuron, and triafamone at three concentration levels of 0.01, 0.1, and 1 mg/L, was loaded to the TLC plates which is equivalent of 0.2, 2, and 20 ng/spot analytical concentrations.…”

“…To separate and identify each herbicide; pyrazosulfuronethyl, pretilachlor, cyhalofop-p-butyl, penoxsulam, ethoxysulfuron, and triafamone standard solution in the range of 0.01-1 mg/L were standardized and validated for precision, linearity, sensitivity and repeatability including intraday, and intraday variations at lower (0.01 mg/L), middle (0.1 mg/L) and higher concertation (1 mg/L) levels using F 254 TLC plates with the desired solvent system along with field samples. TLC method validation was performed following the guidelines set by the European Commission, [32], the International Conference on Harmonization , [33], the International Union of Pure and Applied Chemistry [34], and EURACHEM guidelines, [35] and as followed by Kamal et al [36] and results are presented in Table 2.…”