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”, 3rd edition, Kluwer, 2003Chapters 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”, 3rd edition, Kluwer, 2003Chapters 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 CO2 – hybrid gas-liquid state at 80oC, 10,000 psi– fluid pumped through sample cell, collected at ambient pressure,

60oC and rotary evaporated

Diagram of SFE

From Brazilian J. of Chem. Eng. at http://www.scielo.br/scielo.php?

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 acidsLipid 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)– http://lipidlibrary.aocs.org/topics/silver/index.htm