Procedure

• A stock solution of standard cholesterol was prepared by dissolving cholesterol(0.05 mg/ml) in toluene and sonicated for 10 minutes over an ultrasonic bath.
• Edible oils were mixed with toluene and sonicated for 30 minutes for proper mixing.
• The solution was filtered through Whatman No. 41 filter paper and filtrate was used as sample solution.
• A 20cm × 10cm HPTLC plate coated with silica gel 60 F254 and alumina60 F254 (E. Merck, Darmstadt, Germany) was used for analysis.
• The samples were applied at 10 mm from the base of HPTLC plate by means of a Camag (Switzerland) Linomat V sample applicator using a syringe (100µL, Hamilton, Bonaduz, Switzerland).
• A linear calibration curve was obtained on applying the increasing concentration of standard amino acids in the range (200-1400 ng).
• HPTLC analysis was performed on a computerized densitometer scanner 3, controlled by WinCATS planar chromatography manager version 1.4.4. (CAMAG, Switzerland).
• Plate was developed to a distance of 80 mm, in a Camag twin-trough chamber with mobile phase [n-Hexane: Diethyl ether: MeOH:: 5:2:0.5 (v/v)].
• Plates were evaluated by densitometry at 200 nm with a Camag Scanner 3 for quantification.

Observation

The chromatographic profile of the sample was simple, showing cholesterol as the main component. Peak of cholesterol was identified using the solvent system [n-Hexane: Diethyl ether: MeOH:: 5:2:0.5 (v/v)] with the Rf value of 0.37 ± 0.01 and there was no overlap with any other analyses of the sample at 200 nm (Fig.1).

The linearity of the proposed method was confirmed in the range of 100-700 ng of standard cholesterol. A linear regression of the data points for standard cholesterol is resulted in a calibration curve with the equation Y=644.436 + 652.7908x [regression coefficient (r2) = 0.99831, standard deviation (S.D.) = 2.13%] (Fig. 2). Cholesterol content in edible oils was found to be in the range of 150-710 ng/spot selected for study (Table 1).

Table1: Cholesterol levels in edible oils

The linearity, accuracy in terms of recovery % and precision was considered for the method. Validation of the method at three concentration levels was carried out by the standard recovery formula [9] returned a mean of 90.66%. Precision (repeatability) was determined by running a minimum of four analyses and the coefficient of variability was found to be 1.662 %. The limit of detection (LOD) and quantification (LOQ) was found to be 10 and 32 ng respectively.