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.

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.

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.

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%.

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.