title: Development and validation of TLC-densitometric method for determination of lipid A adjuvant as a bulk and in solid fat nanoemulsions.
abstract: A simple, sensitive, selective and precise high-performance thin-layer chromatographic method was developed for determination of lipid A (MPLA) adjuvant as a bulk and in solid fat nanoemulsions. Chromatographic separations were performed on thin-layer chromatography aluminum plates precoated with silica gel 60 F-254 as stationary phase and chloroform-methanol-ethyl acetate solution (10:2:4, v/v/v) as mobile phase. With this solvent system, compact spots for MPLA at Rf value 0.80 ± 0.02 were obtained. Densitometric analysis of MPLA was carried out in absorbance mode at 357 nm. Linear regression analysis for the calibration plots showed good linear relationship with r = 0.9996 in the concentration range of 20-100 ng/spot. The mean values (±SD) of slope and intercept were found to be 7.355 ± 0.006 and 109.52 ± 0.170, respectively. Limits of detection (LOD) and quantitation (LOQ) were observed at 3.096 and 9.382 ng/spot, respectively.The method was validated for precision, accuracy, robustness and recovery as per the International Conference on Harmonization guidelines. Statistical analysis proved that the developed method for quantification of MPLA as a bulk and in solid fat nanoemulsions is reproducible, selective and economical. This method could be applied for quantitative assay of MPLA in lipid-based vaccine formulations.
authors.affiliation: SLT Institute of Pharmaceutical sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, C.G., 495009, India.
authors.forename: Sunita
authors.initials: S
authors.lastname: Minz
authors.affiliation: SLT Institute of Pharmaceutical sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, C.G., 495009, India.
authors.forename: Monika
authors.initials: M
authors.lastname: Kaurav
authors.affiliation: SLT Institute of Pharmaceutical sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, C.G., 495009, India.
authors.forename: Kantrol Kumar
authors.initials: KK
authors.lastname: Sahu
authors.affiliation: SLT Institute of Pharmaceutical sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, C.G., 495009, India.
authors.forename: Vivekananda
authors.initials: V
authors.lastname: Mandal
authors.affiliation: SLT Institute of Pharmaceutical sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, C.G., 495009, India.
authors.forename: Ravi Shankar
authors.initials: RS
authors.lastname: Pandey
mesh_terms: D002138:Calibration; D002855:Chromatography, Thin Layer / Q000295:instrumentation / Q000379:methods*; D003720:Densitometry / Q000379:methods*; D004655:Emulsions / Q000032:analysis* / Q000737:chemistry; D057230:Limit of Detection; D016014:Linear Models; D008050:Lipid A / Q000032:analysis*; D049329:Nanostructures / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity
chemical_list: D004655:Emulsions; D008050:Lipid A
keywords: high-performance thin-layer chromatography; monophosphoryl lipid A; solid fat nanoemulsions