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.

Fatty acid and sn-2 fatty acid composition in human milk from Granada (Spain) and in infant formulas.

OBJECTIVE: To investigate differences in fatty acid and sn-2 fatty acid composition in colostrum, transitional and mature human milk, and in term infant formulas. SETTING: Departament de Nutrició i Bromatologia, University of Barcelona, Spain and University Hospital of Granada, Spain. SUBJECTS: One-hundred and twenty mothers and 11 available types of infant formulas for term infants. DESIGN: We analysed the fatty acid composition of colostrum (n=40), transitional milk (n=40), mature milk (n=40) and 11 infant formulas. We also analysed the fatty acid composition at sn-2 position in colostrum (n=12), transitional milk (n=12), mature milk (n=12), and the 11 infant formulas. RESULTS: Human milk in Spain had low saturated fatty acids, high monounsaturated fatty acids and high linolenic acid. Infant formulas and mature human milk had similar fatty acid composition. In mature milk, palmitic acid was preferentially esterified at the sn-2 position (86.25%), and oleic and linoleic acids were predominantly esterified at the sn-1,3 positions (12.22 and 22.27%, respectively, in the sn-2 position). In infant formulas, palmitic acid was preferentially esterified at the sn-1,3 positions and oleic and linoleic acids had higher percentages at the sn-2 position than they do in human milk. CONCLUSION: Fatty acid composition of human milk in Spain seems to reflect the Mediterranean dietary habits of mothers. Infant formulas resemble the fatty acid profile of human milk, but the distribution of fatty acids at the sn-2 position is markedly different.

Direct determination by high-performance liquid chromatography of sn-2 monopalmitin after enzymatic lipase hydrolysis.

An alternative method to determine the sn-2 monopalmitin in infant formulas was developed and validated. This method offers many advantages over the traditional methods. It follows the official method in the first steps, purification of the fat or oil through an alumina column, and subsequently the triglycerides are incubated with pancreatic lipase in order to obtain the sn-2 monoglycerides. In traditional methods the sn-2 monoglycerides are separated by preparative thin-layer chromatography and then, the 2-monoglycerides are converted into the corresponding fatty acid methyl esters and analysed by gas chromatography. In our method, separation, quantification and identification of the sn-2 monoglycerides were achieved by high-performance liquid chromatography with evaporative light-scattering detection. The detection limit (0.19 microg), quantification limit (0.38 microg), linearity range (r=0.999, range 1-200 microg) and precision (SD=1.10) show the suitability of the proposed method. This method is faster, cheaper and simple and does not consume large quantities of reagents and materials.

The influence of dietary palmitic acid triacylglyceride position on the fatty acid, calcium and magnesium contents of at term newborn faeces.

The distribution of long-chain saturated fatty acids in triglycerides is different in infant formulas to that in human milk. In human milk, palmitic acid is predominantly esterified in the sn-2 position (beta-position) of the triglycerides, whereas in infant formulas, it is esterified mainly in the sn-1,3 positions (alpha,alpha'-positions). The specific distribution of the fatty acids in the triglyceride plays a key role in lipid digestion and absorption. We studied fatty-acid, calcium and magnesium composition in the faeces of three groups of at term newborn infants fed different diets: Group A (n=12) was fed from birth to 2 months with human milk (66% palmitic acid in beta-position), Group B (n=12) was fed with formula alpha (19% palmitic acid esterified in beta-position) for 2 months, and Group C (n=12) was fed with formula alpha during the first month and with formula beta (44.5% palmitic acid in beta-position) during the second month. Samples were taken at the end of the first month (t0) and at the end of the second month (t1). Groups A and C presented significantly lower contents of palmitic acid in faeces at t1 than at t0, whereas in Group B, amounts remained similar. Faecal calcium in Groups A and C decreased in the second month (t1), although the fall was no statistically significant. In Group B, calcium amounts showed no change. We found that infant formula beta when compared with infant formula alpha reduced significantly the contents of total fatty acids and palmitic acid in faeces. We conclude that palmitic acid in beta-position is, therefore, beneficial for term infants.

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 direct methods for the determination of fatty acids in infant feces.

We have validated and compared two direct methods for the determination of fatty acids in feces by capillary gas chromatography. Method I consisted of esterification of fatty acids using acetyl chloride. Method II used boron trifluoride-methanol as esterification reagent. The two methods were assayed with and without previous freeze-drying of the fecal sample. We found that the two methods could be carried out without sample freeze-drying. Precision and recovery rates were determined and the results were satisfactory. Both methods gave similar results, but Method II has certain advantages over Method I, such as speed, safety, and better recovery rates.

Plasma and erythrocyte alpha-tocopherol and plasma retinol concentrations in term infants fed formula enriched with long-chain polyunsaturated fatty acids.

OBJECTIVE: To assess the effect of long-chain polyunsaturated fatty acids (LCPUFA)- and vitamin E-supplemented formula feeding on erythrocyte and plasma alpha-tocopherol (VE), and plasma retinol (VA) concentrations in neonates and to compare these values with those found in infants feeding on infant formula without LCPUFA or breast milk SETTING: University Hospital of Granada, Spain. SUBJECTS: 49 full-term infants. DESIGN AND INTERVENTION: Subjects who chose not to breast feed were fed either (i) unsupplemented infant formula (F) or (ii) infant formula supplemented with LCPUFA and vitamin E (FL). Alpha-tocopherol and retinol were measured at 7 days, 1 month and 3 months. RESULTS: Plasma and erythrocyte VE concentrations and plasma VE/total lipids ratio increased significantly in all groups at 1 month of life (P < 0.05), but did not change significantly between 1 month and 3 months in any group (P > 0.05). Erythrocyte VE and VA retinol concentrations were higher in infants fed an infant formula than in breast milk-fed infants at 1 month of life (P < 0.05). Finally, there were no significant differences in plasma or erythrocyte VE levels, plasma VA or plasma VE/total lipid ratio between any groups at 3 months of life (P > 0.05). CONCLUSION: Infants fed on LCPUFA- and vitamin E-supplemented infant formula for 3 months have similar vitamin E and A status to infants fed on breast milk or infant formula without LCPUFA supplementation.

Evaluation by high-performance liquid chromatography of the hydrolysis of human milk triacylglycerides during storage at low temperatures.

The effect of various storage methods on the stability of the triacylglyceride fraction of human milk was evaluated. Samples were treated as follows: Group I--stored at -20 degrees C for 4 months, group II--heated for 1.5 min at 80 degrees C and stored at -20 degrees C for 4 months, group III--stored at -80 degrees C for 4 months and thawed rapidly at room temperature (25 degrees C) just before analysis and group IV--stored at -80 degrees C for 2 months, thawed rapidly at room temperature (25 degrees C), then stored at -80 degrees C for a further 2 months and finally thawed rapidly at 25 degrees C just before analysis. The absence of hydrolysis products in group II and group III indicated that these storage procedures were satisfactory even when samples were rapidly thawed for a short time (group IV). Only storage at -20 degrees C without previous heat treatment led to the hydrolysis of triacylglycerides and the appearance of free fatty acids (group I). On the other hand, the effect that freezing and thawing had over the lipolysis grade was studied. Samples were treated as follows: group V--stored at -20 degrees C for 2 months, thawed slowly at refrigerator temperature (5 degrees C), held at this temperature for one week and stored for a further month at -20 degrees C. Freezing and thawing activated lipolysis and increased the production of free fatty acids, monoacylglycerides and diacylglycerides. Milk samples were analyzed by reversed-phase HPLC with a ternary gradient of acetonitrile-dichloromethane-acetone and an evaporative light-scattering detector.

Analysis of human milk triacylglycerols by high-performance liquid chromatography with light-scattering detection.

A high-performance liquid chromatography (HPLC) method for the separation of human milk triacylglycerols using a C18 Spherisorb ODS column and ternary gradient elution with dichloromethane, acetone and acetonitrile is described. The triacylglycerols are detected by light scattering. Several chromatographic conditions were assayed in order to optimize the method: sample solubility, mobile phase, column temperature and the mass detector oven temperature. The linearity, precision and relative response of the method were examined. A total of 34 peaks were separated and quantified based on the percentage peak area in the HPLC chromatogram. Mature human milk analyzed by this method contained six predominant triacylglyceride structures: POO, POL, LOO, POP, OOO and SOP, where P = palmitin, O = olein, L = linolein and S = stearin.

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.

Fatty acid composition of plasma and erythrocytes in term infants fed human milk and formulae with and without docosahexaenoic and arachidonic acids from egg yolk lecithin.

Human milk contains small but nutritionally significant amounts of long-chain polyunsaturated fatty acids (LCP), such as arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids, which are not present in most infant formulae. In the present study, the fatty acid composition of plasma and erythrocytes was determined at birth and again at 7 days, 1 and 3 months in 49 healthy full-term infants (37-42 week's gestation). One group of infants was fed exclusively with human milk (n=16) and the others were randomly assigned to a standard term formula (F) (n=15) or the same formula with egg yolk lecithin providing DHA (0.15%) and AA (0.30%) (LCP-F) (n=18). Plasma and erythrocyte LCP values of the three dietary groups did not differ at 7 days of age, but the contents of DHA and AA in plasma and erythrocytes at 1 and 3 months were significantly lower (P<0.05) in infants fed non supplemented formula than in infants fed breast milk and supplemented formula. There were no differences in plasma or erythrocyte AA or DHA concentrations between the group fed breast milk and the group fed supplemented formula during the period studied.

Influence of caesarean delivery and maternal factors on fat-soluble vitamins in blood from cord and neonates.

We measured plasma and erythrocyte vitamin E (VE) and plasma vitamin A (VA) profiles in 48 full-term and 8 preterm pairs of neonates and their mothers at birth and we determined whether there is any relationship between maternal and umbilical cord for the nutrients measured. At the same time, we assessed the influence of the delivery type and neonate anthropometric measurements on maternal and cord blood VA and VE levels. We measured vitamin levels in vein and arterial blood in order to establish differences due to fetal metabolism. To determine the influence of pregnancy on vitamin levels, we compared the maternal results with data from a group of 13 non-pregnant women. Cord blood had lower plasma VE (arterial 275.8+/-71.7 microg/dl and vein 282.89+/-64.4 microg/dl values), erythrocyte VE (arterial 256.96+/-50.41 microg/dl packet cells and vein 257.41+/-44.35 microg/dl values), and VA levels (arterial 26.72+/-11.83 microg/dl and 27.15+/-10.05 microg/dl values) and a lower vitamin E/total lipids ratio (VE/LT) (arterial 1.60+/-0.4 and vein 1.62+/-0.3 values) than maternal blood (1474.62+/-424.51 microg/dl, 305.94+/-54.75 microg/dl packet cells, 41.03+/-18.83 microg/dl, 2.34+/-0.5, respectively). VA levels were higher in preterm than full-term neonates (P<0.05). Plasma and erythrocyte VE levels were not correlated in maternal blood but were correlated in neonates and infants (r>0.40; P<0.01). We found a good correlation between erythrocyte tocopherol of maternal and cord blood (r>0.40; P<0.01), although there was no correlation with plasma VE values. Cord vein plasma VE levels were higher than cord arterial blood measurements (P<0.01). The plasma VE and VE/LT of the mother and cord following vaginal delivery were higher than measurements from caesarean delivery (P<0.05), although erythrocyte levels were similar. The plasma VE level was higher in mothers at delivery than non-pregnant women.

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.

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 alpha-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 alpha-tocopherol and retinol concentrations did not vary significantly, whereas erythrocyte alpha-tocopherol was significantly higher after the intervention period. CONCLUSION: This study shows that low doses of n-3 PUFA supplemented with adequate amounts of alpha-tocopherol can be incorporated into blood lipids in elderly subjects without lowering their antioxidant concentrations.

Determination of alpha-tocopherol in plasma and erythrocytes by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method for the determination of alpha-tocopherol in plasma or erythrocytes with photodiode-array detection is described. Using this detector, information about the spectrum, absorption maxima and purity of the peak is obtained. Tocopherol was separated on a 5-microns Spherisorb ODS-2 column with methanol as element at a flow-rate of 1.0 ml/min. As little as 100 microliters of plasma or 150 microliters of erythrocytes can be used for accurate analysis with direct extraction without saponification. The speed, specificity, sensitivity and reproducibility of this technique make it particularly suitable for the routine determination of alpha-tocopherol in plasma or erythrocytes.

Determination of sterol content in different food samples by capillary gas chromatography.

An accurate method for the determination of sterols by capillary gas chromatography was developed and applied to the analysis of food. The procedure includes the following steps: dichloromethane-methanol (2:1, v/v) lipid extraction, saponification at 80 degrees C and separation of the unsaponifiable matter with cyclohexane, derivatization to form trimethylsilyl ethers and gas chromatography using 5 alpha-cholestane as the internal standard. The method shows good accuracy, precision and sensitivity and is suitable for the determination of sterols in food.