Development, validation, and application of cholesterol determination method for meat and poultry products using gas chromatography

Abstract

Michael Brown and Joseph Goldstein, two Nobel laureates, declared cholesterol to be the most highly decorated small molecule in biology, with 13 Nobel Prizes awarded to scientists dedicating their professional careers to cholesterol research. Windaus first determined cholesterol with using digitonin in 1910, followed by Grigaut with a colorimetric method using Liebermann-Burchard reaction in 1911. The development of innovative technologies and analytical instruments has allowed for incredible specificity, sensitivity, accuracy, and precision of cholesterol determination. The AOAC Official Method 994.10 employing GC-FID is currently used in most commercial laboratories for routine analysis of cholesterol in foods. Although significantly modified by employing direct saponification, it is still costly, time-consuming, and labor-intensive. The primary objectives of the present study were to refine the analytical conditions for improved performance, develop a written procedure, and determine intra- and inter-laboratory performances of a newly developed method using the AOAC-required performance measurements. Cholesterol was liberated from meat and poultry samples and was detected using GC-FID at a retention time of 11.96 min. Using 0.005 mg of 5α-cholestane/mL as internal standard, the linearity of the standard curve was determined for peak ratio and standard concentrations of 0.008 to 0.020 mg of cholesterol/mL with a coefficient of determination of 0.995 and a response factor of 0.66 (CV = 3.84%). The limits of detection and quantitation were 1.24 and 4.00 mg/100 g, respectively. Cholesterol was stable in the toluene extract when stored for up to 6 d under refrigeration before GC analysis as demonstrated by high precision (0.92 to 2.69% CV) and accuracy (93.24 to 100.56% recovery) of the results. This method was proved to be more accurate and precise compared with results from two commercial laboratories using the AOAC 994.10 method. The new method was also tested for robustness in two ruggedness trials and was collaboratively validated. This method has now been routinely applied to update cholesterol content of meat and poultry in the USDA National Nutrient Database for Standard Reference with high reliability and productivity. Data from 363 meat and poultry samples with 799 replicates showed that 91.5% of total samples were measured with CV of less than 6% and Horwitz ratios of less than 2.0. Collective data for a meat homogenate, SRM1546 (certified reference material), indicated that most of measured concentrations were within the certified range.

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Keywords

Cholesterol, Gas chromatography, Method validation, Meat, Poultry

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