title: Development and validation of a stability-indicating method for determination of free sterols in the Asian medicinal leech Hirudo manillensis.
abstract: A rapid, simple, sensitive, selective, precise and robust thin-layer chromatography densitometric method for the determination of free sterols in leech was developed and validated on silica gel layer using carbon tetrachloride-methanol-formic acid (9.5:1.5:0.55, v/v/v). Spectrodensitometric scanning was carried using a Camag TLC scanner III at 366 nm after spraying 2% methanolic sulphuric acid, which gave compact spots for cholesterol (R(F) = 0.35 ± 0.02). The regression analysis data for calibration plot implied a good linear relationship (r(2) = 0.99958) between response and concentration over the range 100-600 ng per spot with respect to peak area. The limits of detection and quantification were found to be 13.8 ± 0.51 and 45.01 ± 1.29 ng per spot, respectively. Validation was in accordance to the International Conference on Harmonization guidelines. Cholesterol was subjected to forced stress conditions of oxidation, hydrolysis and heat. Degradation products resulting from the forced stress did not interfere with detection because the degradant peaks were well separated from the cholesterol peak. The densitometric method can be regarded as stability-indicating and can be used for quality control assay of cholesterol in leech extract.
authors.affiliation: Department of Pharmaceutics, Hamdard University, New Delhi 110062, India. fatima.hasan@yahoo.com
authors.forename: Fatima
authors.initials: F
authors.lastname: Hasan
authors.forename: Roop K
authors.initials: RK
authors.lastname: Khar
authors.forename: Farhan J
authors.initials: FJ
authors.lastname: Ahmad
authors.forename: Sayeed
authors.initials: S
authors.lastname: Ahmad
authors.forename: Mushir
authors.initials: M
authors.lastname: Ali
authors.forename: Zeenat
authors.initials: Z
authors.lastname: Iqbal
authors.forename: Mohammed H
authors.initials: MH
authors.lastname: Reza
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002251:Carbon Tetrachloride / Q000737:chemistry; D002784:Cholesterol / Q000032:analysis* / Q000737:chemistry*; D002855:Chromatography, Thin Layer / Q000379:methods; D005561:Formates / Q000737:chemistry; D047709:Hirudo medicinalis / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016018:Least-Squares Analysis; D016014:Linear Models; D000432:Methanol / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
chemical_list: D005561:Formates; C030544:formic acid; D002784:Cholesterol; D006861:Hydrogen Peroxide; D002251:Carbon Tetrachloride; D000432:Methanol