Author(s): Kumar S, Monif T, Khuroo A, Reyar S, Verma PRp, Rao S
A selective, accurate, and reproducible novel fourier transform infrared spectroscopy method was developed and validated for quantification of fats in fecal and food sample. Fatty acids were extracted from fecal samples with acidified petroleum ether-ethanol and the extracts were dried and redissolved in chloroform. Quantification was based on the absorbance band of the CH2 group (2855 cm–1) of free fatty acids and fatty acid glycerol esters. The calibration curve of fats in food and fecal matrix showed excellent linearity. The method was proved to be specific, accurate, precise and linear over the concentration ranges of 1.00 g% to 14.98 g% for food and 1.00 g% to 14.96 g% for fecal matrix. The method was validated according to the Guidance for Industry, Bioanalytical Method Validation, US-FDA. The method also showed good performances in terms of accuracy (94.05-101.36% for food matrix and 89.84-103.40% for fecal matrix) and precision (4.9-10.9% for food matrix and 3.1-11.1% for fecal matrix) for fats. The percent recovery of fats ranged from 55.7-60.4% for food matrix and 54.4-70.4% for fecal matrix. Results of all stability studies were within acceptable limits. Incurred sample reanalysis (ISR) was performed by random selection of fecal and food samples. Incurred sample reanalysis data indicated good acceptance and reproducibility of the method and met ISR acceptance criteria, 94% of food and 88% fecal matrix.
The validated bioanalytical method was successfully applied to measure fats concentration in fecal and food matrix samples obtained from clinical study of orlistat capsule, an anti-obesity medication. A total of approximately 1200 fecal and 300 food samples were successfully analyzed. The new FT-IR method is simple, reproducible and enables rapid analysis of fats in clinical samples.