Velimatti Ollilainen

Application of an HPLC assay for the determination of folate derivatives in some vegetables, fruits and berries consumed in Finland

Abstract The folate content of 30 commodities of vegetables, fruits and berries, including a few processed products, was determined by high-performance liquid chromatography (HPLC). HPLC was used with a combination with fluorescence and ultraviolet detectors to analyze folate monoglutamate derivatives and their distribution after extraction at pH 6.0 and deconjugation with hog kidney deconjugase at pH 4.9. 5-Methyltetrahydrofolate was the main derivative in all foods studied, but tetrahydrofolate, 5-formyltetrahydrofolate and 10-formylfolic acid were also detected. The sum of the monitored folate derivatives (as μg folic acid) in vegetables ranged from 9 to 114 μg per 100 g and that of berries and fruits from 3 to 36 μg per 100 g. The variation in folate content, which was studied by analyzing raw potato, carrot and cabbage bought from retail shops three times a year, was small. Some of the studied processed vegetable foods were also reasonably good sources of folate. The results obtained with HPLC are rather similar to the previously reported values for vegetables determined by a microbiological method. Several measures for method improvement and quality control of the analysis allowed reliable determination of the main folate forms, particularly 5-methyltetrahydrofolate, in a wide range of plant-derived foods.

Tocopherols and Tocotrienols in Wheat Genotypes in the HEALTHGRAIN Diversity Screen

Tocopherol and tocotrienol compositions were studied in 175 genotypes of different wheat types grown under similar conditions to screen for natural diversity. The main focus was on bread wheats, including 130 and 20 winter and spring types, respectively. The average total content of tocopherols and tocotrienols was 49.4 microg/g of dm, with a range of 27.6-79.7 microg/g of dm, indicating a 2.9-fold variation among genotypes. Beta-tocotrienol and alpha-tocopherol were the major vitamers, and in general there were more tocotrienols than tocopherols. In the early cultivated forms of wheat the proportion of tocotrienols was especially high, at >or=62.5%. In conclusion, there was a large variation in total tocopherol and tocotrienol contents in bread wheats and this, along with the high proportions of tocotrienols in other types of wheat, demonstrates the great genetic potential of genotypes to be exploited by plant breeders.

Analysis of protein carbonyls in meat products by using the DNPH-method, fluorescence spectroscopy and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS).

Liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) was applied as an advanced methodology to study the suitability of using α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as protein oxidation markers in meat products. The results obtained were compared to those obtained by using the DNPH-method and fluorescence spectroscopy for the analysis of protein carbonyls. Lipid oxidation was also investigated in order to elucidate the relationship between lipid and protein oxidation measurements. Both semialdehydes were originally detected in a food system which proves that lysine, arginine and proline are degraded as a result of oxidative reactions to yield AAS and GGS in meat products. A lack of consistency was observed between the MS results for AAS and GGS and the values obtained by the DNPH-method and the fluorescence spectroscopy. Unlike the last two methods, AAS and GGS measurements have proved to be unaffected by the composition or the structure of the food matrix providing precise information about the fate of particular amino acids during processing of muscle foods. These semialdehydes, and particularly GGS, could be used as indicators of protein oxidation in meat products like TBARS numbers are commonly used as lipid oxidation markers. In fact, a significant correlation was found between GGS values and TBARS highlighting the timely interaction between lipid and protein oxidation.

Simultaneous HPLC analysis of fat-soluble vitamins in selected animal products after small-scale extraction.

Abstract A method for simultaneous determination of fat-soluble vitamins in animal products is presented. Milk, and two fish products containing many ingredients, were used as test materials. The vitamins determined were tocopherols, β-carotene, all- trans -retinol and in the case of fish, cholecalciferol. The sample preparation procedure, consisting of saponification and extraction by n -hexane–ethyl acetate was carried out in small-scale. To cope with the highly different composition of the test materials, some modifications were needed. Normal-phase HPLC separation using diisopropyl ether- n -hexane gradient elution with UV and fluorescence detections for tocopherols, β-carotene and all- trans -retinol was used. Cholecalciferol was determined from the same extract by reverse-phase HPLC (with UV detection) after semi-preparative HPLC purification. Recoveries of spiked samples varied from 80 to 111% for all determined fat-soluble vitamins. The day-to-day repeatability was in most cases under 7%, and the within-day variation of this method was small, under 5.5% for all vitamins. The described analytical method is effective and fast, enabling the processing of a large number of samples.

Analysis of protein oxidation markers α-aminoadipic and γ-glutamic semialdehydes in food proteins using liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS).

To elucidate the formation of protein oxidation biomarkers alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) in food proteins was the main purpose of the present study. Food proteins, namely, myofibrillar proteins, alpha-lactalbumin, and soy proteins, as well as bovine serum albumin (BSA), were suspended in a piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer and oxidized by Fe(3+) and H(2)O(2) while kept in an oven for 14 days at 37 degrees C. For the analysis of semialdehydes, a derivatization procedure with p-aminobenzoic acid (ABA) and NaCNBH(3) followed by liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS) was performed. For comparative purposes, the dinitrophenylhydrazine (DNPH) method was also employed as a routine method to assess carbonyl gain. Both semialdehydes were specifically and accurately detected by LC-MS in all oxidized proteins proving that GGS and AAS are formed as a consequence of the oxidation of lysine, proline, and arginine amino acid residues from BSA and other food proteins. Proteins from an animal source and, particularly, BSA were more susceptible to undergo oxidative reactions than soy proteins. The results from the present paper highlight the significance of using both semialdehydes as protein oxidation indicators in meat and dairy products. The analysis of GGS and AAS in real food systems would contribute to the understanding of the precise mechanisms involved in food protein oxidation and shed light on the fate of oxidizing amino acids during food processing and storage.

Antioxidant Activity of Isolated Ellagitannins from Red Raspberries and Cloudberries

Ellagitannins from red raspberries (Rubus idaeus) and cloudberries (Rubus chamaemorus) were isolated by using column chromatography and preparative HPLC. The berry phenolic isolates consisted of 80% (cloudberry) and of 60% (raspberry) of ellagitannins, with raspberries also containing anthocyanins. The main ellagitannins of both raspberries and cloudberries were identified by ESI-MS to consist of the dimeric sanguiin H-6 and the trimeric lambertianin C. Monomeric ellagitannins such as casuarictin in raspberries and pedunculagin in cloudberries were also found. The antioxidant activity of the berry phenolic isolate, ellagitannin isolate (mixture), ellagitannin main fraction (dimer and trimer), and ellagic acid was studied in bulk and emulsified methyl linoleate, in human low-density lipoprotein in vitro, and the radical scavenging activity was studied in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. Cloudberry and red raspberry ellagitannins were highly effective as radical scavengers. Berry ellagitannins also showed significant antioxidant activity toward oxidation of both human LDL and methyl linoleate emulsions. However, only weak or moderate antioxidant activity was exhibited by ellagitannins toward oxidation of bulk oil. Thus, ellagitannins contribute significantly to the antioxidant capacity of cloudberries and red raspberries in lipoprotein and lipid emulsion environments, the latter being more relevant for food applications.

Acetylcholinesterase inhibitory guided fractionation of Melissa officinalis L.

The plant Melissa officinalis L. has been used traditionally in the treatment of cognitive dysfunction. Based on its traditional medicinal use, it was assessed for its clinical efficacy in mild to moderate Alzheimers patients. The plant was effective in the management of the disease. Therefore, based on this result, a similar plant extract was prepared in order to be screened for bioactivities which are relevant in Alzheimers disease therapy. The extract was recently screened for antioxidant activity and it showed a wide range of antioxidant properties. Another important bioactivity is acetylcholinesterase inhibition, which the extract was screened for in the current investigation. The extract was capable of inhibiting the enzyme in a time and dose-dependent manner. Activity of the extract at 10 min was estimated as 1.72+/-0.16 microg equivalents of physostigmine/mg of the extract. Acetylcholinesterase inhibitory guided fractionation of the extract was then carried out. Most of the fractions showed inhibitory activity and were more potent than the extract. The contents of the most potent fraction were identified as cis- and trans-rosmarinic acid isomers and a rosmarinic acid derivative using LC-DAD-ESI-MS and NMR methods.

Regiospecific determination of short-chain triacylglycerols in butterfat by normal-phase HPLC with on-line electrospray-tandem mass spectrometry.

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C2−C6). The molar correction factors, determined for 42 regioisomeric pairs of short-chain TAG of 20 randomized mixture of standards, differed by 1.4–80% as the ratios varied between 0.217 and 5.847. Butterfat TAG were resolved into four fractions on short flash chromatography grade silica gel columns. Pairs of regioisomers in the TAG classes L/S/S-S/L/S with predominance of L/S/S isomers and the sole regioisomers in the TAG classes L/L(M)/S were identified by tandem MS, where M denotes either 8∶0 or 10∶0 acyl chain. The total proportion of L/L(M)/S isomers was estimated at 34.7 and that of L/S/S-S/L/S at 1.0 mol%, including a small proportion of S/S/S. In contrast to previous work, the present data indicate the presence of a small proportion of butyric and caproic acids in the sn-1-position. The overall distribution of the FA in the short-chain TAG of butterfat, calculated from direct MS measurements, was consistent with the results of indirect determinations based on stereospecific analyses of total butterfat.

Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography, using evaporative light-scattering and electrospray mass spectrometry for detection.

A novel method was developed for the analysis of molecular species in neutral lipid classes, using separation by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 microm-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identification of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour.

Carotenoids and retinoids in finnish foods: Meat and meat products

Abstract The carotenoids and retinoids contained in 33 food items made of meat and meat products were analyzed by high-performance liquid chromatography (HPLC). Compounds were isolated by alkaline hydrolysis and hexane extraction. A nonaqueous reversed-phase column was used to separate carotenoids, and retinoids were determined with a normal-phase silica column. Total β-carotene (all-trans-β-carotene + 15,15′-cis-β-carotene), all-trans-retinol, and its 13-cis-isomer were quantified. The recovery of all-trans-retinol and all-trans-β-carotene added to ground beef (3.4% fat) was 94 and 99%, respectively. The determination limit for each compound was ca. 0.01 μ/g. All-trans-β-carotene and 15,15′-cis-β-carotene, α-carotene, β-cryptoxanthin, all-trans-retinol, and 13-cis-retinol were present in meat and meat products. Retinol and β-carotene were the predominant compounds in beef traces of α-carotene and β-cryptoxanthin were also present. The all-trans-retinol content of beef ranged from 0.03 μg/g (beef top round) to 0.22 μg/ g (beef brisket). The total β-carotene content was 0.22-0.34 μg/g. High β-carotene levels were found in beef liver (8.7 μ/g) and in cow blood (2.7 μ/g). In pork liver, 540 μg all-trans-retinol and 55 μ 13-cis-retinol per 1 g were found. The content of all-trans-retinol in pork ranged from 0.09 to 0.23 μg/g; carotenoids were not found. Mutton, reindeer, venison, and poultry contained mainly retinoids. Both retinoids and carotenoids were present in other meat products, except for ham and bacon, in which only retinoids were detected.