Fats, HPLC analysis

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

High-pressure pumps operating at up to 6000 psi are required to force solvent through a tightly packed HPLC column, and electronic detectors are used to monitor the appearance of material eluting from the column. Alternatively, the column can be interfaced to a mass spectrometer to determine the mass spectrum of every substance as it elutes. Figure 12.18 shows the results of HPLC analysis of a mixture of 10 fat-soluble vitamins on 5 jam silica spheres with acetonitrile as solvent. 

Figure 12.18 Results of an HPLC analysis of a mixture often fat-soluble vitamins.

Burns [27] described a fully automated approach for HPLC analysis of vitamin tablets. A sample valve provides the injection interface in this apphcation. Tablets direct from the production plant are dispensed into the sample cups on a Technicon Sohd Prep sampler, they are dissolved, and the fat-soluble vitamins are extracted. The solution is concentrated... 

The development of HPLC techniqne led to an increase in the number of scientific papers dealing with phenolic evalnation in foods and, in the meantime, it also improved the nnmber and type of foods in which phenolic snbstances were evalnated. Despite the high number of scientific papers, HPLC analysis of phenolic snbstances, except for wine and tocopherols in fats and oils, has never became an official method of analysis, so the nnmber of scientific papers has ever more increased. 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

With samples containing a wide range of components, such as hydrogenated fats, the column was eluted with solvent A for 13 min, then changed in one step to A-B (75 25), with a gradient to 100% B over 20 min. The free fatty acids were converted into the phenacyl derivatives and, prior to HPLC analysis, were purified by elution from a BOND ELUT NH2 column with hexane-diethyl ether (9 1). 

They studied the effect of the mass detectors drift tube temperature on the low-molecular-mass TGs. Solutions of 10 mg/ml of tributyrin, tricaproin, tricaprylin, tricaprin, and trilaurin were injected twice at each of the following drift tube temperatures 20,25, 30,45, and 60°C. Five replications of the HPLC analysis were performed for one sample of ewe s milk fat to determine the reproducibility of the HPLC method. The TG composition was estimated in accordance with the method based on the calculation of the equivalent carbon numbers (ECNs) of the HPLC chromatographic peaks and in the molar composition in fatty acids, analyzed by GLC, collected at the HPLC chromatograph outlet. The HPLC fractions were collected every 40 s at the outlet of the column after 14 min there were no peaks before that time. 

R Andreoli, M Careri, P Manini, G Mori, M Musci. HPLC Analysis of fat-soluble vitamins on standard and narrow bore columns with UV, electrochemical and particle beam MS detection. Chro-matographia 44 605-612, 1997. 

A method capable of quantifying SPC in raw bovine milk was developed. In this procedure the sample was centrifuged at -4°C and the top fat layer removed. The defatted milk was depro-teinated with TCA, and the supernatant was washed sequentially with dichloromethane, hexane, and ethyl acetate. An aliquot of the separated aqueous layer was prepared for the HPLC analysis by mixing with DSA and filtering. The analyte was quantified with an electrochemical detector. Recoveries achieved were 76-80% (116). 

If fat-soluble vitamins (A, D and E) are added to a product, the first step in the analysis will often be hydrolysis. In fortified products this converts any esters, for example, vitamin A palmitate or vitamin E acetate, to their free alcohols. The product is then extracted with a hydrophobic solvent, such as hexane or diethyl ethyl or a mixture of these solvents, prior to HPLC analysis often using a reverse-phase (Ci8) column. 

In this procedure, the HPLC analysis is carried out on a DuPont Zorbax ODS column using methanol, acetonitrile and water 10 2 1 at the mobile phase. This system resolves vitamins D3 from D2 and from degradation products and other fat soluble vitamins. Didecyl and dinonyl phthalate were used as the internal standards. The latter was included for use in the event that extraneous peaks interfered with the didecyl phthalate peak. Figure 11 shows a chromatogram of an extract of a multivitamin formulation with the two added internal standards. 

Figure 6. HPLC analysis of mammary tissue extracts. (A) Rat fed low fat diet containing 0.3% BHT for 2 weeks. (B) Control rat fed low fat diet only. The arrow shows the position of the BHT peak.

Some methods which do not involve separation of the FFAs from the milk fat or the whole product have considerable appeal because of their simplicity. Sharma and Bindal (1987) exploited the property of methyl urea to complex triglycerides in producing methyl esters with BF3-methanol without first separating the FFAs from the fat, while Spangelo et al. (1986) were able to methylate FFAs in an acetonitrile extract of milk with methyl iodide in the presence of an anion exchange resin as catalyst. Miwa and Yamamoto (1990) derivatised the FFAs in milk and milk products for HPLC analysis by direct reaction with 2-nitrophenylhydrazine hydrochloride. 

Triacylglycerol profiles, determined by high-performance liquid chromatography (HPLC), may also be a tool for the detection of the adulteration of pork by beef fat (44). The presence of 5% or more of pork fat in beef or mutton tallow can be detected and quantified by HPLC analysis of fatty acids in the sn-2 position of the triacylglycerols, because the ratio of 16 0/18 loo9 at this position is about 5.0... 

Antioxidants are used widely in fats and oils products to delay oxidative processes. Synthetic antioxidants, namely, butylated hydroxyanisole (BHA), butylated hydro-xytoluene (BHT), tert-butyUiydroquinone (TBHQ), and propyl gallate (PG), are permitted antioxidants that are frequently used in products. Their presence and concentration may be determined with HPLC and GC methods. Meanwhile, metal chelators such as citric acid may be determined by HPLC analysis. 

During malaxation, when water is added to olive paste to wash it, tons of waste water (800.000 in Italy only) are produced from the oil industry and discarded. Water soluble compounds are then transfered from olives into waste waters and could be recovered. HPLC analysis of the waste water extract showed the presence of hydroxytyrosol, tyrosol, p-OH benzoic acid, vanillic acid, caffeic acid, oleuropein, verbascoside and other oleuropein derivatives. The extract had powerful antioxidant activity and could represent a cheap source of natural antioxidants to be used to preserve food and cosmetics from fat oxidation and rancidity [51]. 

Papadoyannis, I.N. Tsioni, G.K. Samanidou, V.F. Simultaneous determination of nine water and fat soluble vitamins after SPE separation and RP-HPLC analysis in pharmaceutical preparations and biological fluids. J. Liq. Chromatogr. 1997, 20 (19), 3203-3231. 

Salo-Vaananen, P, Ollilainen, V., Mattila, P., Lehikoinen, K., Salmela-Molsa, E., andPiironen, V. 2000. Simultaneous HPLC Analysis of Fat-Soluble Vitamins in Selected Animal Products after Small-Scale Extraction. Food Chem. 71 535-543. 

The sample preparation and FIPLC analysis are more elaborate for formulations with multiple APIs (e.g., over-the-counter (OTC) products) or with natural products. Examples of HPLC analysis of two OTC multi-vitamin products are shown in Figure 6.5, with a summary of method performance for both water-soluble and fat-soluble vitamins17 listed in Table 6.5. Other examples of HPLC analysis of extracts of natural products (white and red ginseng)18 are... 

HPLC analysis of phospholipids by evaporative light-scattering detection T. L. Mounts, S. L. Abidi, and K. A. Rennick, J. AOCS 69 438 (1992). Determination of cholesterol in milk fat by reversed-phase high-performance liquid chromatography and... 

Although most Ag-HPLC analyses have traditionally been done using CLA methyl esters, improved separation and quantitation of CLA isomers has been noted when phenacyl or p-methoxyphenacyl ester rather than methyl ester derivatives were analyzed (58). Nikolova-Damyanova et al. (59) demonstrated improved separation of the p-methoxyphenacyl derivatives of the cis/trans 8,10- through 11,13-18 2 isomers in a commercial sample of CLA when compared with CLAME. Only a single Ag-HPLC column was required. Resolution of the cis/ trans isomers was similar to that obtained for CLAME using Ag-HPLC systems with two or three Ag-HPLC columns connected in series (60). A stepwise solvent gradient of 100% hexane/dichloromethane/ACN (40 60 0.2 vol/vol/vol 30 min) to 100% dichloromethane/ACN (100 1) over 10 min with UV detection at 270 nm (for phenacyl esters) yielded a semiquantitative estimation of fatty acid composition comparable to, but more detailed than that obtainable by GC analysis as FAME. This method was also applied to detamine the CLA isomer composition of a sample of beef/pig fat, but prefractionation by RP-HPLC was required before Ag-HPLC analysis. The prefractionation step was required to concentrate the CLA isomers and to remove octadecenoate and other fatty acids that might interfere with Ag-HPLC analysis. 

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