M.C. López-Sabater

The effects of harvest and extraction methods on the antioxidant content (phenolics, α-tocopherol, and β-carotene) in virgin olive oil

We studied the effects of harvesting and two processing systems (two-phase centrifugation and three-phase centrifugation) on olive oil quality. Oils extracted from high quality olives do not differ in free acidity, peroxide value and ultraviolet light absorption. Nor was the fatty acid composition affected. However, the antioxidant content of the oil was higher from green olives than from ripe olives. On the other hand, we determined that neither extraction method affects the presence of α-tocopherol and β-carotene, however, the phenolic content is higher in the two-phase method due to the addition of lukewarm water that is used to dilute the olive paste.

Rapid determination of vitamin E in vegetable oils by reversed-phase high-performance liquid chromatography

A quick and direct method for measuring tocopherols (alpha, beta+gamma and delta) in vegetable oils has been developed using RP-HPLC with UV detection. Previous extraction of tocopherols is not required. The oil is diluted in hexane and an aliquot is mixed with ethanol containing an internal standard (alpha-tocopherol acetate). The chromatographic system consists of an ODS-2 column with a methanol-water mobile phase. Tocopherols are detected at 292 nm in less than 5 min after injection. The method is precise (RSD=2.69%) and has a high mean recovery (98.14%).

Effect of ingestion of virgin olive oil on human low-density lipoprotein composition

Objective: To measure the incorporation of oleic acid and antioxidants (phenols and vitamin E) to low density lipoprotein (LDL) after acute and short-term ingestion of virgin olive oil. To study whether this incorporation contributes to an increase in LDL resistance to oxidation.Setting: Department of Food and Nutrition, University of Barcelona, Spain and Department of Lipids and Cardiovascular Epidemiology, IMIM, Barcelona, Spain.Subjects: Sixteen healthy volunteers aged 25–65 y.Design and interventions: To observe the change in the fatty acid profile, vitamin E, phenolic compounds and LDL oxidation-related variables after the postprandial phase and after daily ingestion of olive oil for one week.Results: Few changes were observed in the postprandial phase. However, after a week of olive oil consumption there was an increase in oleic acid (P=0.015), vitamin E (P=0.047), phenolics (P=0.021) and lag time (P=0.000), and a decrease in the maximum amount of dienes (P=0.045) and oxidation rate (P=0.05).Conclusion: After ingestion of virgin olive oil, an increase in antioxidants and oleic acid in LDL was observed as well as an improvement of LDL resistance to oxidation. Our results support the idea that daily ingestion of virgin olive oil could protect LDL from oxidation.Sponsorship: This study was supported by a research grant from Spain (ALI 97-1607-C02-02).

Simultaneous determination of α-tocopherol and β-carotene in olive oil by reversed-phase high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographic method was developed for the determination, in one run, of α-tocopherol and β-carotene in virgin olive oil. The method involved a rapid saponification and a later extraction with a mixture of hexane–ethyl acetate. The chromatographic system consists of an ODS-2 column with a mobile phase of methanol–water–butanol and a diode-array detector. Linearity, precision, recovery and sensitivity were satisfactory. The main advantage of the proposed method is the speed and simultaneous determination of both compounds at the same time.

Determination of inulin in meat products by high-performance liquid chromatography with refractive index detection.

Inulin is a naturally occurring carbohydrate with beneficial nutritional and technological properties. A high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of these beta-fructans in meat products, containing this type of additive. The method includes extraction of inulin with hot water, followed by hydrolysis with inulinase enzyme, and determination of the released fructose by HPLC with refractive index detection. An internal standard of rhamnose was used to quantify fructose. The method incorporates a sample blank (without inulinase hydrolysis) for each specimen to subtract contributions of free fructose and fructose from sucrose. The results showed good precision with average RSDs of 2.4% for repeatability and 5.2% for reproducibility. Analytical recovery ranged from 102 to 106%. Satisfactory linearity (r=0.999) was obtained.

Comparison of two methods for the determination of fatty acid profiles in plasma and erythrocytes

We have validated and compared two methods for the determination of fatty acid profiles in biological samples by capillary gas chromatography. Method I consisted of a previous lipid extraction and esterification of fatty acids using boron trifluoride-methanol. Method II was a direct method that combined extraction and esterification of freeze-dried samples in a single step, using acetyl chloride as the reagent. The two methods were applied to the analysis of plasma and erythrocyte samples. Both methods gave similar results in plasma, whereas in erythrocytes, the direct method gave significantly higher contents of total fatty acids. Precision and recovery rates were determined and the results were satisfactory. Detection and quantification limits showed that both methods had excellent sensitivity. It was concluded that the direct method has substantial advantages over the conventional method, such as higher values in erythrocytes, rapidity and less possibility of contamination or fatty acid losses. Therefore, it is preferable for the analysis of biological samples such as plasma and erythrocytes.

Rapid high-performance liquid chromatographic method for the simultaneous determination of retinol, α-tocopherol and β-carotene in human plasma and low-density lipoproteins

A reversed-phase HPLC method with diode-array detection was used to simultaneously determine retinol, α-tocopherol and β-carotene in human plasma and low-density lipoproteins. An aliquot of sample was de-proteinized with ethanol containing α-tocopherol acetate as internal standard, and the analytes were extracted twice with hexane. The solvent was evaporated to dryness under a stream of nitrogen and the residue was redissolved in methanol to be injected directly into the HPLC system. A multiple solvent system based on methanol, butanol and water at a flow-rate of 2 ml/min and held at 45°C provided clear separation of these compounds in only 8 min. The method showed good linearity, precision and accuracy for all compounds. Owing to its simplicity, this method may be useful in routine clinical and epidemiological work.

High-performance liquid chromatography with evaporative light-scattering detection for the determination of phospholipid classes in human milk, infant formulas and phospholipid sources of long-chain polyunsaturated fatty acids ☆

We developed and validated a new high-performance liquid chromatographic method for the separation of phospholipid classes in human milk, infant formulas and phospholipidic sources of long-chain polyunsaturated fatty acids (LC-PUFAs) used in paediatric nutrition. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were separated in less than 25 min using an Extrasil silica column (150 x 4.0 mm I.D., 3-microm particle size) by isocratic elution with a mixture of isopropanol-hexane-water. Phospholipids were determined by an evaporative light-scattering detector. Several chromatographic conditions were assayed to optimise the method, whose suitability is shown by the detection limits, linearity ranges and precision rates obtained. The main advantages of the proposed method are its speed and the direct determination of the main phospholipids present in human milk, infant formulas and the phospholipid sources of LC-PUFAs used in paediatric nutrition.

Comparison of two direct methods for the determination of fatty acids in human milk

SummaryWe have validated and compared two direct methods for determining by capillary gas chromatography fatty acid composition in human colostrum and one method that involved lipid extraction and then methylation. Direct Method 1 (DM1) consisted of transesterification of fatty acids using acetyl chloride. Direct Method 2 (DM2) used boron trifluoride-methanol as esterification reagent. The two direct methods were compared with a traditional methylation method that involves prior lipid extraction. We found that there were no differences between the direct methods and the traditional method. We then validated both direct methods; recovery rates, inter-assay and intra-assay precisions were determined with satisfactory results. Both methods gave similar results, but DM2 has certain advantages over DM1, such as speed, safety and better recovery rates.

Administration of low doses of fish oil derived N-3 fatty acids to elderly subjects

Objectives: To assess the incorporation of n-3 polyunsaturated fatty acids (PUFA) in plasma and erythrocyte lipids of elderly subjects after ingestion of very low doses of fish oil. The effects on α-tocopherol and retinol concentrations were also studied. Setting: Municipal nursing home in Barcelona, Spain. Subjects: Forty-five elderly subjects aged 60–92 y. Design and intervention: Subjects received a non-commercialized milk formula containing 1% fish oil for 15 months, which provided 0.40 g/d of n-3 PUFA. Fatty acid profiles and antioxidant concentrations were measured before and after the intervention period. Results: Fish oil ingestion was associated with significant increases in total n-3 PUFA in plasma and erythrocytes by 32% and 18%, respectively. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid concentrations were higher after the ingestion period both in plasma and erythrocytes (P<0.05), whereas linoleic and arachidonic acids remained unchanged. The n-6/n-3 ratio decreased by 21% in plasma and by 16% in erythrocytes (P<0.05). Moreover, younger subjects showed a greater incorporation of EPA and DHA than older subjects. Plasma α-tocopherol and retinol concentrations did not vary significantly, whereas erythrocyte α-tocopherol was significantly higher after the intervention period. Conclusion: This study shows that low doses of n-3 PUFA supplemented with adequate amounts of α-tocopherol can be incorporated into blood lipids in elderly subjects without lowering their antioxidant concentrations. Sponsorship: This study was supported by a research grant from the Spanish CDTI.