Extended Validation of a Simplified Extraction and Gravimetric Determination of Total Fat to Selected Foods

Abstract

A simplified method for chloroform/methanol extraction and gravimetric determination of total fat previously published for diet composites was tested on additional foods, including milk, cheese, fried snack foods, nuts/seeds, salad dressings, baked goods, meat, fish/shellfish and fruits/vegetables. Homogenized food composites and certified reference materials (CRMs) were analyzed using the existing method involving, briefly, orbital shaking of a subsample with chloroform/methanol/buffer in specific proportions, then recovery of total lipid in the chloroform extract. For meat, fish, shrimp, cheese and fried plantains, total fat recovery was low and/or variability was high with the unmodified assay versus results from other standard total fat methods (e.g., ether extraction, acid hydrolysis) or to CRM assigned values. Reducing the sample size or adding a homogenization step during extraction resulted in relative standard deviations of <3% for all matrices and also values consistent with those generated by other standard total fat methods and within assigned ranges for total fat in most CRMs. PRACTICAL APPLICATIONS The method allows for routine batch analysis of multiple samples of a range of food types with minimized hands‐on time, using commonly available laboratory equipment. The resulting total lipid extract can be subdivided for analysis of various specific lipid analytes as well as for gravimetric measurement of total fat.

Optimization of Standard Gas Chromatographic Methodology for the Determination of Trans Fat in Unlabeled Bakery Products

“…Initial clear establishment that C18:1-12t and C18:1-13t might be unresolved from C18:1-9c at higher concentrations (e.g., those specified in AOAC 996.06) in high-trans samples Lipid extraction Total lipid extraction (Phillips et al 2008) instead of the homogenization/acid or alkaline digestion step and extraction with diethyl ether/petroleum ether Previous validation of method (Phillips et al 2008) and specific testing/validation on the proposed food matrices, and conformity with existing laboratory standard operating procedures Methylation 30 min, instead of 45 min saponification; 5 ml saturated sodium chloride instead of 5 ml water and 1 g sodium sulfate; two 2 ml iso-octane extractions of FAME with concentration to 1 ml under nitrogen at 40°C instead of one 1 ml hexane extraction Previous validation of method and conformity with established laboratory analytical standard operating procedures (Phillips et al 2008) Retention time standards Some of the standards in AOAC 996.06 are listed as unspecified isomers (octadecanoic acid) and all three conjugated linoleic acid (CLA) isomers, but for RT determination, C18:1-13c, C18:1-13t, and other CLAs (more than the three unspecified CLAs in AOAC 996.06) are used. Also, the C22:1 cetoleic standard was not used to determine the RT, only the C22:1-13c (erucic acid).…”

“…The final volume was adjusted if necessary to ∼1.0 ml with iso-octane, then the Validation of methodology to optimize GC based on previously established linear range and calibration, as previously reported (Phillips et al 1997). Methodology for quantitative lipid extraction previously established (Phillips et al 2008), making addition of IS before extraction unnecessary and a portion of extract is taken for gravimetric total lipid analysis before addition of IS for fatty acid analysis Total lipid in sample extract analyzed for fatty acids…”

“…Total lipid was extracted from 0.8 to 6.0 g of thawed homogenized sample (ensuring that the analytical portion was a representative subsample) using a chloroform/methanol extraction as reported previously and validated for bread/cake/cereal matrices (Phillips et al 2008). A portion of the total lipid extract estimated to contain 20-30 mg total lipid (typically 10 ml) was precisely measured using a Hamilton Microlab 900 dispenser (Fisher Scientific) into a 15-ml test tube containing 1.2 mg of the C11:0 triglyceride internal standard (IS; 2.0 ml of a 0.6-mg/ ml solution in chloroform).…”

“…Previous analytical experience (Phillips et al 2008) dictated the selection of the compounds to be quantified extract was transferred into a GC crimp seal vial(s) with 100 µl insert(s), blanketed with nitrogen, sealed, and then analyzed by GC. FAME were separated and quantified by GC with flame ionization detection using a Perkin Elmer Autosystem™ GC and PE Nelson Turbochrom™ 4 software (Perkin Elmer, Waltham, MA) and a 100 m×0.25 mm column with 0.2 µm 100% nonbonded bis-cyanopropyl polysiloxane film (Supelco SP2560, Cat.…”

Preparation and characterization of control materials for the analysis of conjugated linoleic acid and trans-vaccenic acid in beef

“…A stock beef lipid solution was prepared by extracting total lipid from twelve portions of 1.2 g homogenized beef fat (~89% fat) using previously reported methodology (Phillips, Ruggio, & Amanna, 2008) and combining 50 mL (2 × 25 mL) precisely measured portions of each extract, by dispensing the solution into 50 mL glass test tubes using a Hamilton Microlab 900 (Fisher Scientific, Waltham, MA), to yield a total of~8.0 g fat (12 × 1.2 g × 89% fat in the beef fat samples × 50 mL/ 80 mL = 8.0 g). The extracts were concentrated to 10-15 mL each under nitrogen at 40°C, then quantitatively transferred with chloroform to one 200 mL volumetric flask and brought to volume with chloroform, yielding a total lipid concentration of~40 mg/mL.…”