Erich Grundel

HPLC analysis for trans-vitamin K1 and dihydro-vitamin K1 in margarines and margarine-like products using the C30 stationary phase

Abstract A C 30 column successfully separated the cis and trans isomers of vitamin K 1 in margarines, reduced-fat margarine-like products and in their ingredient oils. We also measured the compound 2′3′-dihydro-vitamin K 1 , a derivative formed during hydrogenation of oils containing vitamin K 1 . We compared an enzymatic procedure, currently under AOAC collaborative study for milk and infant formulas, with a more direct extraction method in analyzing margarines and margarine-like products. Both methods have good precision and were applicable to the majority of products examined. Margarines or margarine-like products identifying liquid soy-bean oil, hydrogenated soybean oil or liquid canola oil as their primary ingredients contained about 50–160 μg vitamin K 1 /100 g. Blends of sunflower and soybean oils contained 1 /100 g. Hardened or “stick” margarines contained more 2′3′-dihydro-vitamin K 1 than “soft” or “tub” products (122–285 μg/100 g vs 38–131 μg/100 g, respectively). Eight of 18 products (44%) contained 10% or more of the Reference Daily Intake for vitamin K 1 per serving. Higher-fat margarines contained more vitamin K 1 than their lower-fat counterparts.

Determination of selected biogenic amines in Acacia rigidula plant materials and dietary supplements using LC–MS/MS methods

Extracts of Acacia rigidula leaves are used in weight-loss products sold in vitamin shops and over the internet with little or no published data about their potential biological effects. In our chemical investigations on authenticated A. rigidula plant material, we established a rapid and sensitive LC-MS/MS method for the quantitative determination of several phenethylamine, tyramine and tryptamine derivatives. Stable isotopically labeled compounds were used as internal standards for quantitative analysis. We found total calculated contents of 6 biogenic amines in A. rigidula leaf of 18.6 and 32.9μg/g. The content of selected amines in 21 dietary supplements labeled as containing A. rigidula was determined by a second LC-MS/MS method. Our study revealed significant differences in the amine profiles of authenticated plant materials and dietary supplements. β-Methylphenethylamine, a non-natural compound, was found in 9 of the 21 dietary supplement products. β-Methylphenethylamine was found at levels of 960-60,500μg/g while phenethylamine was found at levels of 710-171,620μg/g. β-Methylphenethylamine is a positional isomer of amphetamine and our results showed that it can be misidentified as amphetamine during LC-MS analysis. An independent GC-MS analysis was used to confirm the presence of β-methylphenethylamine and the absence of amphetamine in dietary supplements labeled as containing A. rigidula. This study demonstrates that confirmations by independent analytical methods are essential to verify findings of unusual or unexpected compounds in dietary supplements.

Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates

Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity. In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity. Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSH-DHP) which were quantified using LC-MS-MS analysis. Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.

Vegetable concentrates interact with canthaxanthin to affect carotenoid bioavailability and superoxide dismutase activity but not immune response in rats

Abstract We examined tomato paste and dried spinach powder as dietary sources of lycopene and lutein and determined their interactions with canthaxanthin (CX) in water-soluble beadlets. Mature male rats, 10/group, were fed a basal diet containing 16% fat and 2 g per kg CX from beadlets (+CX) or placebo beadlets (−CX) for 8 weeks. Tomato paste or spinach powder was added to each of these diets at 0, 5 (low tomato, low spinach) and 15% (ww) (high tomato, high Spinach). The low and high levels of tomato paste and spinach powder contained 0.03 and 0.09 g lycopene and 0.02 and 0.06 g lutein per kg of diet, respectively. CX was detected in liver and plasma. High tomato decreased liver CX concentrations 5-fold and plasma CX 2-fold; low tomato had no effect. Liver lycopene concentrations increased as the concentration of tomato paste increased in the diet. However, feeding CX dramatically decreased liver lycopene concentrations. Feeding high tomato and no CX lowered liver superoxide dismutase activity. Neither dietary carotenoids nor CX treatment altered mitogenic response of splenic mononuclear cells to concanavalin (Con A) or lipopolysaccharide (LPS). These data from rats illustrate how a carotenoid-rich food may influence the bioavailability of a carotenoid supplement. Likewise, supplementation with a single purified carotenoid may antagonize the bioavailability of carotenoids in food matrices.

Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicum

Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate-based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl-t-butyl ether (90:10), both containing 0.1% diethylamine.

Reduced fat diets influence α-tocopherol concentrations in plasma lipoproteins and tissues of male and female rats

Abstract This study examined the effects of feeding low-fat diets containing comparable levels of vitamin E and a fat source rich in unsaturated fatty acids (sunflower oil blend). Weanling male and female Sprague Dawley rats were used in the study. Three groups of each gender (15 rats/group) were fed, for six weeks, diets containing 5.6% fat or 2.8% fat in which the dietary fat was replaced by cornstarch or a commercially available fat-free margarine product. The diets were a modification of the AIN-76 diet. Food intake was significantly increased in male and female rats fed the reduced fat diet containing the fat-free margarine. Caloric intakes and body and tissue weights of the animals were not affected by the dietary treatments. Rats fed the reduced-fat diets had higher concentrations of plasma α-tocopherol, liver α-tocopherol, and plasma HDL α-tocopherol than did rats fed the 5.6% fat diet. Female rats receiving the reduced-fat diet containing the fat-free margarine had significantly lower concentrations of total plasma and HDL α-tocopherol compared with the group fed the diet in which the fat was replaced by cornstarch. Reductions in the fat level of diets rich in unsaturated fatty acids can enhance vitamin E transport and storage when dietary vitamin E levels are maintained. The magnitude of the changes in α-tocopherol metabolism in the rat depends on gender and the dietary components used as a substitute for the fat.