HPLC for Food Analysis

1 HPLCfor FoodAnalysisA Primer Copyright Agilent Technologies Company, 1996-2001. All rights reserved. Reproduction, adaption, or translationwithout prior written permission is prohibited, except asallowed under the copyright in Germany September 01, 2001 Publication Number FoodAnalysisThe fundamentals of analternative approach tosolving tomorrow smeasurementchallengesAngelikaGratzfeld- H sgen andRainer SchusterA PrimerAcknowledgementsWe would like to thank ChristineMiller and John Jaskowiak fortheir contributions to this Miller is an applicationchemist with Agilent Technologiesand is responsible for the material contained in chapter 5.

2 Mr. Jaskowiak, who wrote chapter 7,is a product manager for liquidchromatography products atAgilent Technologies. Copyright Agilent Technologies Company 1996-2001. All rights reserved. Reproduction,adaption, or translation without priorwritten permission is prohibited, exceptas allowed under the copyright in Germany, September 1, Number 5988-3294 ENIIIP refaceModern agriculture and food processing often involve theuse of chemicals. Some of these chemicals and their func-tions are listed below: Fertilizers: increase production of agricultural plants Pesticides: protect crops against weeds and pests Antibiotics: prevent bacteria growth in animals duringbreeding Hormones: accelerate animal growth Colorants: increase acceptability and appeal of food Preservatives and antioxidants: extend product life Natural and artificial sweeteners and flavors: improvethe taste of food Natural and synthetic vitamins.

3 Increase the nutritivevalue of food Carbohydrates: act as food bindersSuch chemicals improve productivity and thus increasecompetitiveness and profit margins. However, if theamounts consumed exceed certain limits, some of thesechemicals may prove harmful to countries therefore have established official tolerancelevels for chemical additives, residues and contaminants infood products. These regulations must be monitored care-fully to ensure that the additives do not exceed the pre-scribed levels. To ensure compliance with these regulatoryrequirements, analytical methods have been developed todetermine the nature and concentration of chemicals infood products.

4 Monitoring of foodstuffs includes a check of both the raw materials and the end product. To protectconsumers, public control agencies also analyze selectedfood liquid chromatography (HPLC) is usedincreasingly in the Analysis of food samples to separate anddetect additives and contaminants. This method breaksdown complex mixtures into individual compounds, whichin turn are identified and quantified by suitable detectorsand data handling systems. Because separation and detec-tion occur at or slightly above ambient temperature, thismethod is ideally suited for compounds of limited thermalstability.

5 The ability to inject large sample amounts (up to1 2 ml per injection) makes HPLC a very sensitive analysistechnique. HPLC and the nondestructive detection tech-niques also enable the collection of fractions for furtheranalysis. In addition, modern sample preparation tech-niques such as solid-phase extraction and supercritical fluidextraction (SFE) permit high-sensitivity HPLC Analysis inthe ppt (parts per trillion) range. The different detectiontechniques enable not only highly sensitive but also highlyselective Analysis of 1 Match of analyte characteristics to carrier mediumHPLCH ydrophobicPolarityHPLCGCV olatileNonvolatileVolatilityVolatile carboxylic acidsNitrilesNitrosamineEssential oilsOrgano- phosphorous pesticidesGlyphosateAlcoholAromatic estersPCBI norganic ionsAldehydes KetonesBHT, BHA.

6 THBQ antioxidantsPAHsHydrophilicSulfonamidesE poxidesTMS derivative of sugarsC2/C6 hydrocarbonsFatty acidmethylesterPolymer monomersGlycolsAromatic aminesAnabolicaFat soluble vitaminsTriglyceridesNatural food dyesPG, OG, DG phenolsAmino acidsSynthetic food dyesFatty acidsSugarsSugaralcoholsFlavonoidsAntibi oticsEnzymesAflatoxinsPhospho-lipidsIts selective detectors, together with its ability to connect amass spectrometer (MS) for peak identification, make gaschromatography (GC) the most popular separates and detects at ambient temperatures.

7 Forthis reason, agencies such as the Food and DrugAdministration (FDA) have adopted and recommendedHPLC for the Analysis of thermally labile, nonvolatile, highlypolar electrophoresis (CE) is a relatively new but rap-idly growing separation technique. It is not yet used in theroutine Analysis of food, however. Originally CE was appliedprimarily in the Analysis of biological macromolecules, butit also has been used to separate amino acids, chiral drugs,vitamins, pesticides, inorganic ions, organic acids, dyes, , 2, 3 Part 1 is a catalog of analyses of compounds in foods.

8 Eachsection features individual chromatograms and suggestsappropriate HPLC equipment. In addition, we list chromato-graphic parameters as well as the performance characteris-tics that you can expect using the methods shown. In part 2we examine sample preparation and explain the principlesbehind the operation of each part of an HPLC system sam-pling systems, pumps, and detectors as well as instrumentcontrol and data evaluation stations. In the last of 11 chap-ters, we discuss the performance criteria for HPLC, whichare critical for obtaining reliable and accurate results.

9 Part 3contains a bibliography and an 1 Analytical examples of food additivesAcidulants .. 2 Antioxidants .. 4 Preservatives .. 6 Artificial sweeteners .. 8 Colorants .. 10 Flavors .. 12 Vanillin .. 12 Bitter compounds: hesperidin and naringenin .. 14 Chapter 2 Analytical examples of residues andcontaminantsResidues of chemotherapeutics and antiparasitic drugs .. 16 Tetracyclines .. 18 Fumonisins .. 19 Mycotoxins .. 21 Bisphenol A diglydidyl-ether (BADGE) .. 24 Pesticides .. 26 Carbamates.

10 28 Glyphosate .. 29 Chapter 3 Analytical examples of naturalcomponentsInorganic anions .. 32 Lipids .. 35 Triglycerides and hydroperoxides in oils .. 35 Triglycerides in olive oil .. 37 Fatty acids .. 38 Carbohydrates .. 40 Vitamins .. 42 Water-soluble vitamins .. 42 Fat-soluble vitamins .. 45 Analysis of tocopherols on normal-phase column .. 46 Biogenic amines .. 48 Amino acids .. 50 Peptides .. 52 VIPart OneThe HPLC ApproachChapter 4 separation in the liquid phaseSeparation mechanisms.