Food Composition Analysis Moisture and Total Solids – Part 1 Ash – Part 1 Protein Analysis – Part 1 Vitamin Analysis (Discussed separately in Vitamins and coenzymes slides) Lipid (Fat) Analysis – Part 2 Carbohydrate Analysis – Part 3 Secondary Metabolites and Nutraceuticals (Analysis methods to be discussed as we go) From: Nielsen, “Food Analysis”, 3 rd edition, Kluwer, 2003 Chapters 8 and 14.

Lipids Mainly present in foods as triacylglycerols (fats and oils), other fatty acid esters, phospholipids (e.g. lecithin), sterols and other isoprenoids, fat- soluble vitamins (A, D, E, K) Lipid composition varies greatly between animal and plant-based foods and within plant-based foods (table 8-2) Most lipids are soluble in nonpolar solvents (e.g. ether, chloroform) but some also have limited water-solubility From: Nielsen, “Food Analysis”, 3 rd edition, Kluwer, 2003 Chapters 8 and 14.

Lipids – quantitative analysis Accurate lipid analysis requires effective extraction from the food matrix extraction methods tailored to particular food nonpolar solvents utilized – hexane, ethyl ether, petroleum ether extract, dry and weigh hydrolysis may be needed with complex lipids to release lipid from carbohydrate/protein moieties steps may be taken to minimize oxidation –addition of BHT or other antioxidant

Extraction of lipids Most effective if samples pre-dried Samples are ground to reduce particle size, maximize surface area Blending helps saturate particles Hydrolysis (1 hr reflux w 3 M HCl or saponification with ethanolic KOH) needed with dairy, grains, animal products Sequential solvent extractions of plant materials begin with least polar solvent and increase in polarity so that lipids are removed first, other constituents follow Reverse-phase separation methods isolate the polar constituents first, lipids last

Lipid extraction methods Continuous solvent extraction – sample exposed to boiling solvent for several hrs Semicontinuous (Soxhlet extraction) – same idea but periodic solvent exposure Mojonnier flask or Babcock methods (dairy) use a digestion step prior to allowing fat to separate Microwave-assisted extraction often used w dairy, meats Ultrasound-assisted extraction Supercritical fluid extraction – “green” method uses pressurized CO 2 –hybrid gas-liquid state at 80 o C, 10,000 psi –fluid pumped through sample cell, collected at ambient pressure, 60 o C and rotary evaporated

Diagram of SFE From Brazilian J. of Chem. Eng. at

Instrumental lipid quantification NMR – low resolution is effective because one monitors broader regions –CW NMR can determine degree of unsaturation –pulsed NMR – oil vs. solid fat content can be determined - protons relax faster in solids –signal amplitude is proportional to quantity IR - fat absorbs strongly at C=O stretching region (1745 cm -1 ) –meat, dairy, oilseeds x-ray absorption – higher in lean meat than fat ultrasound – fat content in meats

Lipid characterization Ch. 14 (Nielsen) Iodine value – degree of unsaturation Saponification equivalents Free fatty acids Lipid oxidation status & oxidation products TBARS test for malondialdehyde conjugated dienes & trienes by UV volatile organics by GC fatty acid composition by GC (14.6.2) trans fatty acids by IR (14.6.3) – total trans fatty acids quantified based on trans C=C absorption at 967 cm-1

Chemistry of TBARS test Colored adduct is measured at 530 nm

From Methods of Analysis for Functional Foods and Nutraceuticals, Chapter 2” Fatty acids can be derivatized to their volatile methyl esters (FAME) acid/MeOH or NaOMe can be used short chain FA are volatile, some water solubility; can prepare iPr esters instead derivatization as DMOX (4,4-dimethyloxazolines) often used GC-MS employed if standards are inadequate Capillary GC on moderately polar PEG or carbowax, medium length column (25 m x 0.25 mm) separates most FAME Longer (100 m) cyanopropyl columns needed to separate trans-FA from cis-isomers complex mixtures of FAME including  -3/  -6 fatty acids can be separated by cap-GC in < 30 min (Fig. 2.3). GC analysis for FA composition

Cap GC analysis of fish/sunflower oil mixture

Conjugated linoleic acid (CLA) Discussed in detail in Hurst, Ch. 2 Analysis poses a challenge because of the mixture of isomers Cap GC on 100 m cyanopropyl column requires long runtime (80 min) Silver-ion HPLC: Ag + forms polar pi-bonded complexes with unsat. FA –Si column packings impregnated with silver developed for lipid analysis (normal-phase LC) –