Determination of the cardiac drug amiodarone and its N-desethyl metabolite in sludge samples.

For the first time, a procedure for the simultaneous determination of the iodinated drug amiodarone and its major metabolite, N-desethylamiodarone, in sludge from urban sewage treatment plants (STPs) is proposed. Matrix solid-phase dispersion (MSPD) followed by on-line cationic exchange clean-up, in modular configuration, was used as sample preparation technique. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, was employed for the selective determination of target compounds. The optimized procedure provided exhaustive recoveries with little effect of the sample matrix in the efficiency of electrospray ionization (ESI). The overall recoveries of the method ranged between 95 and 111%, for samples spiked at different concentration levels. The achieved limits of quantification (LOQs) remained below 10ngg(-1) for both compounds, and the linear response range extended up to 2500ngg(-1). Amiodarone and N-desethylamiodarone were ubiquitous in sludge samples, from different STPs located in the Northwest of Spain, with maximum concentrations above 300ngg(-1) referred to the freeze-dried matrix. They were also present in stabilized sludge (mixed with lime and thermally dehydrated), which is mostly disposed in agriculture fields as fertilizer. Furthermore, mono-iodinated analogues of amiodarone and N-desethylamiodarone were also tentatively identified in some samples from their accurate MS and MS/MS spectra.

Fast determination of virgin olive oil phenolic metabolites in human high-density lipoproteins.

In recent years it has been confirmed that the consumption of olive oil prevents the oxidation of biomolecules owing to its monounsaturated fatty acids (MUFA) and phenolic content. The main objective of the study was to develop an ultra-high-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the determination of phenolic compounds in human high-density lipoprotein (HDL) samples. At the same time, the influence of olive oil consumption on the phenolic metabolite levels was evaluated in a European population. The participants were 51 healthy men, aged 20-60. They were randomized to two consecutive intervention periods with the administration of raw olive oil with low and high polyphenolic content. The UHPLC-MS/MS analytical method has been validated for hydroxytyrosol and homovanillic acid in terms of linearity (r(2) = 0.99 and 1.00), repeatability (5.7 and 6.5%) reproducibility (6.2 and 7%), recovery (98 to 97%), limits of detection (1.7 to 1.8 ppb) and quantification (5.8 and 6.3 ppb).The levels of the studied metabolites increased significantly after high polyphenolic content virgin olive oil ingestion (p <0.05) compared with lowpolyphenolic content olive oil. Virgin olive oil consumption increases the levels of phenolic metabolites in HDL and thus provides human HDL with more efficient antioxidant protection.

Fatty-acid composition of maternal and umbilical cord plasma and early childhood atopic eczema in a Spanish cohort.

BACKGROUND/OBJECTIVES: Fatty-acid status during in-utero development might influence the risk of atopic diseases in early childhood. The aim of this work was to identify the relationship between maternal plasma and cord blood fatty acid (FA) composition and the risk of atopic eczema in the offspring at 14 months of age. SUBJECTS/METHODS: Two hundred and eleven non-atopic mothers and their children were studied. Mothers were recruited in their first trimester of gestation and children were monitored until 14 months of age. Samples of maternal plasma and cord blood plasma were analyzed to determine the FA profile of total lipids. Presence of atopic eczema in the infants was documented through questionnaires at 6 and 14 months of age. RESULTS: Higher concentrations of total long-chain polyunsaturated FA (LC-PUFA) were found in maternal plasma of non-atopic children in relation to atopic group. Moreover, this maternal plasma LC-PUFA content was negatively correlated with the atopic eczema (odds ratios (OR)=0.83, P=0.04) in infants. Regarding cord blood samples, docosahexaenoic acid (DHA C22:6n3) and the sum of total n-3 and of LC-PUFA n-3 showed a negative correlation with the prevalence of the disease (OR=0.50, 0.49 and 0.49, respectively). CONCLUSIONS: Our results show that the fatty-acid status of the fetus during pregnancy has an important role in the development of atopic eczema in early childhood. The prevalence of this atopic disorder is related to lower cord blood plasma levels of FA belonging to n-3 series, especially DHA.

Effects of prolonged breastfeeding and colostrum fatty acids on allergic manifestations and infections in infancy.

BACKGROUND: In developed countries World Health Organization recommendation of 6 months' exclusive breastfeeding is under debate. OBJECTIVE: We assessed the impact of predominant breastfeeding (PBF) duration and colostrum long-chain polyunsaturated fatty acids (LC-PUFAs) profile on the risk of allergic manifestations (wheezing and atopic eczema) and infections [low respiratory tract infections (LRTIs) and gastroenteritis] in infancy. METHODS: Information on child feeding practices was obtained from 580 infants of a pregnancy cohort. Presence of infant's health outcomes was documented through questionnaires at 6 and 14 months of age. The LC-PUFAs were measured in colostrum. Adjusted odds ratios (adjOR) and 95% confidence intervals (CI) were estimated using multivariable logistic regression models. RESULTS: In comparison with never breastfeeding, PBF for 4-6 months was associated with lower risk of wheezing (adjOR = 0.53; 95% CI, 0.32, 0.89), LRTIs (adjOR = 0.51; 95% CI, 0.31, 0.83) and atopic eczema (adjOR = 0.58; 95% CI, 0.32, 1.04) between months 7 and 14 of life. Results of a risk period-specific analysis (restricted to infants at risk for outcome onset after 6 months of age), showed no indication for reverse causation (results were not very different compared with an overall analysis). Predominantly breastfeeding for 4-6 months was associated with lower risk of gastroenteritis during the first 6 months of life (adjOR = 0.34; 95% CI, 0.18, 0.64). Among breastfed infants higher doses of arachidonic acid (AA), docosahexaenoic acid, and total n-3 in were associated with a decreased risk of gastroenteritis, but no association was found for allergic manifestations or LRTI. CONCLUSIONS AND CLINICAL RELEVANCE: Promotion of PBF for 4-6 months could reduce the burden of allergic manifestations and infections in infancy. Beneficial effects of breastfeeding on gastroenteritis were explained in part by exposure to higher doses of n-3 and AA received from colostrum. No significant effects from fatty acid dose were found on risk of allergic manifestations or LRTIs.

Effects of cooling and freezing storage on the stability of bioactive factors in human colostrum.

Breast milk constitutes the best form of newborn alimentation because of its nutritional and immunological properties. Banked human milk is stored at low temperature, which may produce losses of some bioactive milk components. During lactation, colostrum provides the requirements of the newborn during the first days of life. The aim of this study was to evaluate the effect of cooling storage at 4°C and freezing storage at -20°C and -80°C on bioactive factors in human colostrum. For this purpose, the content of IgA, growth factors such as epidermal growth factor, transforming growth factor (TGF)-ß1 and TGF-ß2, and some cytokines such as IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α, and its type I receptor TNF-RI, were quantified. Some colostrum samples were stored for 6, 12, 24, and 48 h at 4°C and others were frozen at -20°C or -80°C for 6 and 12 mo. We quantified IgA, epidermal growth factor, TGF-ß1, and TGF-ß2 by indirect ELISA. Concentrations of IL-6, IL-10, and TNF-α cytokines, IL-8 chemokine, and TNF-RI were measured using the BD Cytometric Bead Array (BD Biosciences, Erembodegem, Belgium). Bioactive immunological factors measured in this study were retained in colostrum after cooling storage at 4°C for at least 48h, with the exception of IL-10. None of the initial bioactive factor concentrations was modified after 6 mo of freezing storage at either -20°C or -80°C. However, freezing storage of colostrum at -20°C and -80°C for 12 mo produced a decrease in the concentrations of IgA, IL-8, and TGF-ß1. In summary, colostrum can be stored at 4°C for up to 48 h or at -20°C or -80°C for at least 6 mo without losing its immunological properties. Future studies are necessary to develop quality assurance guidelines for the storage of colostrum in human milk banks, and to focus not only on the microbiological safety but also on the maintenance of the immunological properties of colostrum.

Maintenance of breast milk Immunoglobulin A after high-pressure processing.

Human milk is considered the optimal nutritional source for infants. Banked human milk is processed using low-temperature, long-time pasteurization, which assures microbial safety but involves heat denaturation of some desirable milk components such as IgA. High-pressure processing technology, the subject of the current research, has shown minimal destruction of food macromolecules. The objective of this study was to investigate the influence of pressure treatments on IgA content. Moreover, bacterial load was evaluated after pressure treatments. The effects of high-pressure processing on milk IgA content were compared with those of low-temperature, long-time pasteurization. Mature human milk samples were heat treated at 62.5 degrees C for 30min or pressure processed at 400, 500, or 600MPa for 5min at 12 degrees C. An indirect ELISA was used to measure IgA in human milk whey obtained after centrifugation at 800xg for 10min at 4 degrees C. All 3 high-pressure treatments were as effective as low-temperature, long-time pasteurization in reducing the bacterial population of the human milk samples studied. After human milk pressure processing at 400MPa, 100% of IgA content was preserved in milk whey, whereas only 72% was retained in pasteurized milk whey. The higher pressure conditions of 500 and 600MPa produced IgA retention of 87.9 and 69.3%, respectively. These results indicate that high-pressure processing at 400MPa for 5min at 12 degrees C maintains the immunological protective capacity associated with IgA antibodies. This preliminary study suggests that high-pressure processing may be a promising alternative to pasteurization in human milk banking.

Effects of dietary fibre intake on risk factors for cardiovascular disease in subjects at high risk.

BACKGROUND: Epidemiological studies and feeding trials with supplements suggest that fibre intake is associated with a reduction in cardiovascular risk. However, the effects of changes in dietary fibre on risk factor levels have not been evaluated in free-living individuals. Thus, the effects of changes in dietary fibre intake on cardiovascular risk factors were assessed over 3 months in free-living high-risk subjects. METHODS: 772 high-risk subjects (age 69+/-5 years) were assigned to a low-fat diet or two Mediterranean-style diets. All participants received behavioural and nutritional education, including recommendations for increasing the consumption of vegetables, fruits, and legumes. Changes in food and nutrient intake, body weight, blood pressure, lipid profiles, glucose control and inflammatory markers were evaluated. RESULTS: Most participants increased consumption of vegetable products, but the increase in dietary fibre exhibited wide between-subject variability (6-65 g/day). Body weight, waist circumference, and mean systolic and diastolic blood pressure decreased across quintiles of fibre intake (p<0.005; all). Reductions in fasting glucose and total cholesterol levels, and increments in HDL cholesterol were highest among participants in the upper 20% of fibre intake (p = 0.04 and 0.02 respectively). Plasma concentrations of C-reactive protein, but not those of inflammatory cytokines, decreased in parallel with increasing dietary fibre (p = 0.04). Significant reductions in LDL cholesterol were observed only among participants with the greatest increases in soluble fibre intake (p = 0.04). CONCLUSIONS: Increasing dietary fibre intake with natural foods is associated with reductions in classical and novel cardiovascular risk factors in a high-risk cohort.

[Study of maternal nutrition and genetic on the foetal adiposity programming (The PREOBE study)]. / Estudio de la influencia de la nutrición y genética maternas sobre la programación del desarrollo del tejido adiposo fetal (Estudio PREOBE).

BACKGROUND: Maternal genetics and feeding before and during pregnancy, different maternal metabolic pathologies, as well as nutrient intakes of newborns in their first months of life may be involved in the obesity aetiology and its long-term consequences. The possible role of these and others factors, the mechanisms and the effects on the metabolism, and the development of this disease need further research. OBJECTIVE: To acquire more knowledge about foetal adipose tissue development and the influence of genetic, dietetic and environmental factors on the risk to suffer from obesity. METHODOLOGY: Four study groups have been established with 30 pregnant women in each one: 1) control group; 2) mothers with glucose intolerance/gestational diabetes; 3) women with low weight gain during pregnancy, and 4) women with overweight/obesity at the beginning of the pregnancy. The magnitudes to be studied are: 1) dietary intake; 2) life-style habits; 3) physical activity; 4) anthropometry and body composition; 5) haematological study; 6) biochemical study (lipid and metabolic biomarkers); 7) immune function profile related to nutritional status; 8) psychological profile; 9) genetic biomarkers, and 10) microbiological markers; all of them in relation to the development of the foetal adipose tissue in the first stages of life and the risk of suffering from obesity in the future. CONCLUSION: This project, coordinated by the Department of Paediatrics of the School of Medicine in the University of Granada, and with the collaboration of well-known and expert research groups, tries to contribute to the knowledge about the obesity aetiology in infancy and its subsequent development in later periods of life.

Anti-inflammatory effect of virgin olive oil in stable coronary disease patients: a randomized, crossover, controlled trial.

OBJECTIVES: To assess the effect of two similar olive oils, but with differences in their phenolic compounds (powerful antioxidant compounds), on inflammatory markers in stable coronary heart disease patients. DESIGN: Placebo-controlled, crossover, randomized trial. SETTING: Cardiology Department of Hospital del Mar and Institut Municipal d'Investigació Mèdica (Barcelona). SUBJECTS: Twenty-eight stable coronary heart disease patients. INTERVENTIONS: A raw daily dose of 50 ml of virgin and refined olive oil (ROO) was sequentially administered over two periods of 3-weeks, preceded by 2-week washout periods in which ROO was used. RESULTS: Interleukin-6 (P<0.002) and C-reactive protein (P=0.024) decreased after virgin olive oil intervention. No changes were observed in soluble intercellular and vascular adhesion molecules, glucose and lipid profile. CONCLUSIONS: Consumption of virgin olive oil, could provide beneficial effects in stable coronary heart disease patients as an additional intervention to the pharmacological treatment.

Determination of conjugated linoleic acid in human plasma by fast gas chromatography.

A new method for the determination of the main isomers of conjugated linoleic acid (CLA) in human and animal plasma was developed by gas chromatography coupled to flame ionization detection (GC-FID). The new method introduces three main advantages in comparison to the current available methodologies: firstly it does not require previous lipid extraction, secondly the chromatographic separation of CLA isomers was performed on an Rtx-2330 column significantly shorter and thinner than the typical long highly polar capillary columns in use that allows a faster analysis than in current methodologies, and thirdly the amount of sample needed to perform the analyses was substantially lower than the amount used in current routine methodologies. Its application to human plasma and rat plasma showed to be robust and reliable for quick and correct identification of the main CLA isomers in particular, and the total fatty acid profile in general, in routine analysis.

Compliance with the European and national nutritional objectives in a Mediterranean population.

OBJECTIVE: To analyze compliance with the current European and Spanish nutritional objectives in a representative sample from Catalonia, a Spanish Mediterranean region; and to examine relationships between diet and plasma fatty acid composition. DESIGN: Cross-sectional nutritional survey. SETTING: Population based random sample derived from the Catalan Nutrition Survey. SUBJECTS: A total of 516 healthy adult men (n=203) and women (n=313). METHODS: Dietary habits were assessed by means of a quantitative food frequency questionnaire. A physical exam included height, weight, waist and hip circumferences, and a fasting blood draw. RESULTS: Gender differences were observed in nutrient and energy intakes. Women showed a better compliance with the nutritional recommendations for monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) than did men. Men showed a better compliance for saturated fatty acid (SFA) and carbohydrate than did women. However, the SFA:MUFA:PUFA ratio was similar in both gender (1.6:2.3:1.0 for men; 1.7:2.5:1.0 for women). The highest compliance was observed for nutritional goals of sodium, calcium and fruit and vegetable intakes for both genders. In addition, the present study showed that levels of certain fatty acids in plasma are clearly associated with dietary intake of foods rich in these components. The highest correlations were found for n-3 long chain polyunsaturated fatty acids with blue fish intake in both men and women (r (men)=0.36 and r (women)=0.42; P<0.001). CONCLUSIONS: The diet followed in Catalonia seems to ensure compliance with most of the intermediate nutritional objectives for the Spanish population. However, a reduction in the SFA intake and an increase in the carbohydrate intake could be recommended in order to reduce the current prevalence of overweight and obesity in this Mediterranean region. SPONSORSHIP: This study was supported by the Catalan Department of Health, the Nutrition Catalan Centre of the Institute of Catalan Studies, and Mercadona SA.

Determination of phospholipid fatty acids in biological samples by solid-phase extraction and fast gas chromatography.

A new method for the determination of phospholipid fatty acids in biological samples, combination of solid-phase extraction (SPE) and fast gas chromatography (GC) was developed. Its application to human plasma and human erythrocytes showed to be robust and reliable for quick and correct identification in routine analysis.

Determination of gamma- and alpha-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV-vis detection and comparison with evaporative light scattering detection.

A rapid direct method (Method I) for measuring gamma- and alpha-tocopherols in human milk was developed and validated using reversed-phase high-performance liquid chromatography with ultraviolet/visible (UV-vis) detection. Human milk, with an internal standard (alpha-tocopherol acetate) added, was diluted in hexane. The chromatographic system consisted of a short column (50 mm x 2.1mm I.D., 3 microm particle size) that allowed the separation of the gamma- and alpha-tocopherols in less than 6 min. The new direct method (Method I) was compared with other methods. Method II (saponification with ultraviolet/visible detection) determined 24% and 22% less gamma- and alpha-tocopherols, respectively. Method III (saponification with evaporative light scattering detection) gave the same values for alpha-tocopherol content as Method II. However, the amount of sample used in the application of Method III was higher than that used in Method II. Furthermore, Method I uses smaller amounts of solvents, and it is simpler and faster than Methods II or III. Only a small volume of sample is needed, which is an additional advantage for biological assays.

Influence of dietary source of docosahexaenoic and arachidonic acids on their incorporation into membrane phospholipids of red blood cells in term infants.

Here we studied whether the chemical structure of dietary arachidonic and docosahexaenoic acids in full-term infant diets affects their incorporation into erythrocyte membrane phospholipids. From birth to 3 months, infants were fed breast milk (n = 9) or formula milk containing arachidonic acid and docosahexaenoic acid provided by egg phospholipids (n = 10) or by low-eicosapentaenoic acid fish oil and fungal triglycerides (n = 10). We compared the fatty acid composition of erythrocyte phosphatidylethanolamine, phosphatidylcholine and sphingomyelin before and after administration of the experimental diet. At 3 months, infants on formula milk showed lower concentrations of docosahexaenoic acid (in phosphatidylcholine and in phosphatidylethanolamine) and arachidonic acid (in phosphatidylcholine) than those receiving breast milk. We conclude the incorporation of the two fatty acids into erythrocyte phospholipids depends mainly on the lipid composition of the diet received rather than the chemical form in which they are delivered.

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method.

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.

Antioxidant effect of virgin olive oil in patients with stable coronary heart disease: a randomized, crossover, controlled, clinical trial.

The Mediterranean diet, in which olive oil is the main source of fat, has been associated with a reduced incidence of coronary heart disease (CHD) and low blood pressure levels. Virgin olive oil (VOO), besides containing monounsaturated fat, is rich in phenolic compounds (PC) with antioxidant properties. The aim of this study was to examine the antioxidant and anti-hypertensive effect of two similar olive oils, but with differences in their PC (refined: 14.7 mg/kg versus virgin: 161.0 mg/kg), in 40 males with stable CHD. The study was a placebo controlled, crossover, randomized trial. A raw daily dose of 50 mL of VOO and refined olive oil (ROO) were sequentially administered over two periods of 3 weeks, preceded by 2-week washout periods in which ROO was used. Lower plasma oxidized LDL (p < 0.001) and lipid peroxide levels (p = 0.003), together with higher activities of glutathione peroxidase (p = 0.033), were observed after VOO intervention. Systolic blood pressure decreased after intake of VOO (p = 0.001) in hypertensive patients. No changes were observed in diastolic blood pressure, glucose, lipids, and antibodies against oxidized LDL. Consumption of VOO, rich in PC, could provide beneficial effects in CHD patients as an additional and complementary intervention to the pharmacological treatment.

Headspace gas chromatographic method for determining volatile compounds in infant formulas.

Powder infant milk formula quality deterioration and consequently the termination of shelf life results in the appearance of off-flavors mainly determined by a composite effect of spoilage volatiles. A headspace gas chromatographic method to determine propanal, pentanal and hexanal as the main volatiles present in the headspace of powder infant formula oxidation is described as a rapid indication of oxidative status. Under optimum conditions the limits of detection for propanal, pentanal and hexanal were 17.19, 16.87 and 19.60 ng and the limits of quantification were 37.37, 31.96 and 35.97, respectively. The calibration graphs of the method were linear from 25 to 1500, 20 to 3500 and 30 to 8500 ng for propanal, pentanal and hexanal, respectively, with determination coefficients exceeding 0.99. The precision results showed that the relative standard deviations of the repeatability and reproducibility were between 2.2 and 5.5%. The analytical method was simple, rapid, and reliable and permitted the analysis of a large number of formulas using small sample volumes.

Triacylglycerol markers of mature human milk.

OBJECTIVES: To determine which triacylglycerol (TAG) species in mature human milk are less affected by external factors and may thus be considered as TAG markers, as well as to determine which species are most influenced by these external conditions. Furthermore, we examine the correlation between the TAG markers and their fatty acids (FAs). SETTING AND SUBJECTS: Six healthy women from Barcelona (Catalonia, Spain). DESIGN AND INTERVENTIONS: In order to obtain the maximum variability of sampling conditions, 40 mature human milk samples were collected from different mothers, on different days, at different times of the day, and from different breasts during and after both the baby's and mother's meal. TAG and FA profiles were determined and correlated. The TAG composition was determined by high-performance liquid chromatography with an evaporative light-scattering detector, and also with atmospheric pressure chemical ionisation mass spectrometry. FAs compositions were determined by gas chromatography. RESULTS: The results were analysed using the SPSS statistical package and proved to be more variable than might have been found in a more restrictive sample design. Nevertheless, despite these conditions, some TAG species were found in relatively constant levels in mature human milk, and could thus be considered as markers of the mature milk TAG profile. TAG species that we can classify in this group were: LaMO, CaPO, LaCaO, LaPCa, LaOL, MPLn, LLO, LaOO, MPL, and MOL. The names do not indicate the location of fatty acids in the glycerol molecule. On the other hand, concentrations of other TAG species vary considerably between samples and consequently these may be understood to be especially affected by the external factors. TAGs like PaLS, MPO, PaOO, PPP, MPS, SPP, LOO, PPO, MOS, SSP, POL, and SOS are in this second group. Correlation between the TAG markers and their FAs was examined by Pearson's test and a significant correlation was found for some FAs. CONCLUSIONS: The TAG species present in mature human milk are affected in different ways by external factors such as dietary intake, nutritional status, length of lactation, time of the day, etc. Some TAGs may be considered as markers of mature human milk as they are relatively constant under a wide range of sampling conditions and do not depend on the factors mentioned. SPONSORSHIP: This study was supported by the Fundació Mestres Jané.

Rapid determination by reversed-phase high-performance liquid chromatography of Vitamins A and E in infant formulas.

A rapid, sensitive method has been developed for the simultaneous determination of retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate. We compare two experimental procedures for simultaneous direct solvent extraction of these vitamins without previous saponification. Method I: the fat milk sample was extracted with ethanol-hexane and injected directly into the chromatographic column. Method II: the power milk sample was extracted with ethanol-hexane and also injected directly into the column. Under optimum conditions the limits of detection for retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate were 0.33, 21.2, 32.9, 32.5 and 3.2 ng and the limits of quantification were 0.42, 25.3, 37.9, 36.8 and 6.3 ng, respectively. The precision results showed that the relative standard deviations of repeatability and reproducibility were between 0.74 and 5.7%.

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.