Determination of niacin in infant formula by solid-phase extraction and anion-exchange liquid chromatography.

A peer-verified, solid-phase extraction (SPE)/anion exchange liquid chromatographic method is presented for the determination of niacin in milk-based and soy-based infant formula. Analysis is in 3 steps: test sample digestion, extraction/cleanup, and liquid chromatography (LC). Digestion uses a standard AOAC digestion procedure that involves autoclaving at 121 degrees C for 45 min in (1 + 1) H2SO4 to free endogenous niacin from protein and to convert added niacinamide to niacin. The digest solution is adjusted to pH 6.5 with 7.5M NaOH. Acidification to pH <1.0 with (1 + 1) H2SO4 precipitates the protein. The clarified solution is then filtered, and the filtrate is brought to volume. SPE of niacin is accomplished by passing an aliquot of the digest solution through an aromatic sulfonic acid-SPE (ArSCX-SPE) column. After the column is washed with methanol and water to remove extraneous material, the niacin is eluted with 0.25M sodium acetate/acetic acid buffer at pH 5.6. An anion-exchange polystyrene-divinylbenzene column with 0.1 M sodium acetate/acetic acid buffer at pH 4.0 is used for LC. Niacin is determined by UV detection at 260 nm. A standard curve is prepared by passing known amounts of niacin through the ArSCX-SPE columns used for niacin extraction. The following values for x and relative standard deviation (RSD) were obtained for National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula with a certified value for niacin of 63.3 +/- 7.6 microg/g: Submitting laboratory.-- x = 59.7 +/- 4.0 microg/g; RSD = >6.7%; confidence interval (CI) = +/- 1.4 microg/g; n = 27. Peer laboratory.--x = 56.6 +/- 6.6 microg/g; RSD = >11.7%; CI =+/- 4.1 microg/g; n = 8.

Determination of niacin in infant formula and wheat flour by anion-exchange liquid chromatography with solid-phase extraction cleanup.

Niacin content must be included on food labels of infant formula products and bakery products containing enriched flour. Liquid chromatographic (LC) determination of niacin in complex food matrixes is complicated by the presence of endogenous compounds that absorb at the commonly used wave-length of 260 nm. Also, the presence of particulate matter in the standard sulfuric acid extraction procedure results in reduced life of LC columns and precolumns. A simple, rapid, solid-phase extraction (SPE) procedure for separation and cleanup of niacin from a complex food matrix digest has been developed. By using a vacuum manifold with the SPE column system, multiple samples can be processed quickly and efficiently for LC analysis, compared with gravimetric column cleanup. Sulfuric acid sample digest is passed over an aromatic sulfonic acid cation-exchange (ArSCX-SPE) or a sulfonated Florisil SPE column. Niacin is eluted with 0.25M sodium acetate-acetic acid, pH 5.6 buffer in vacuo. LC chromatograms of the resulting eluate are free of interference from other components absorbing at 260 nm at the retention time of niacin. Validation of the method was obtained from agreement of analytical results on available reference materials. For both SPE methods, values for niacin in SRM 1846 Infant Formula (milk-based powder) were within uncertainty ranges of the certified value. Use of several calibration procedures (the LC computer program, a peak area response graphic standard curve, or the method of standard additions) with both SPE procedures resulted in niacin values for 3 RM-Wheat Flours (not certified for niacin) in agreement (90-105%) with their respective values reported in the literature. Several commercial wheat flours showed a broad 260 nm interference, resulting in high niacin values. Niacin recoveries from spiked soy-based liquid infant formulas ranged from 95-107% with the ArSCX-SPE column. Calibration curves of niacin were linear up to 400 micrograms/mL, with a detection limit of 0.2 microgram/mL.

Utilization of chromatographic and spectroscopic techniques to study the oxidation kinetics of selenomethionine.

Ion-exchange LC and spectroscopic supporting techniques have been successfully used to study the kinetics and mechanism of oxidation reactions of selenomethionine (SeMet). Oxidation of selenomethionine with both cyanogen bromide (CNBr) and hydrogen peroxide (H(2)O(2)) proceeds through a stable intermediate which undergoes cyclization and C-Se bond cleavage to form 2-amino-4-butyrolactone. This stable intermediate was identified by IR spectroscopy as methionine dihydroxy selenide. The CH(3)-Se moiety of SeMet formed methyl selenic acid upon reaction with H(2)O(2) and methyl selenocyanate (CH(3)SeCN), characterized by GC-MS, for the reaction with CNBr. Both reactions were of apparent first order with respect to the concentration of SeMet. A rate constant (k(1))of 4.0x10(-3) s(-1) for the reaction of SeMet with HO and 4.0x10(-3) s(-1) for the reaction with CNBr were determined at a temperature of 22 degrees C. Oxidation of methionine (Met) gives disparate kinetics and oxidation products from SeMet. Thus the differential rate method can be utilized to quantitatively separate SeMet in biological samples in the presence of much higher concentrations of Met.

Determination of Iodide in Milk Using the Iodide Specific Ion Electrode and its Application to Market Milk Samples.

The iodide specific ion electrode was used to measure the iodide content of raw and commercially processed whole and skim milks. Average iodide values for raw milk and commercially processed milks were 220 and 620 µ g/L, respectively. The specific ion electrode can also be used to measure iodide in milk contaminated with iodophor sanitizing agent since the milk converts iodophor titratable iodine to the ionic iodide. Since free sulhydryl groups are also detected by the iodide electrode, the effects of heating milk on free sulfhydryl formation and electrode activity were established. These data indicate that in conventionally pasteurized milk, sulfhydryl groups are non-reactive and are not detected by the iodide electrode. However, the increase in free sulfhydryl formation was reflected by an increase in iodide electrode activity at temperatures above those of pasteurization. Whereas the iodide specific ion electrode has been previously used successfully to measure iodide content of raw milk, this method has now been shown to be applicable to pasteurized milk if the conventional pasteurization time-temperature relationship of accepted public health standards is not exceeded.

Minerals in whey and whey fractions.

Liquid and dried acid and sweet wheys and the concentrates and permeates obtained from ultrafiltration of whey were analyzed for major and trace minerals. Calcium, magnesium, sodium, and potassium were determined by atomic absorption and phosphorus by a colorimetric method. Zinc, iron, copper, and manganese were determined by flameless atomic absorption. The composition of all wheys and their fractions differed in nutritionally important minerals. Calcium was three times as great and zinc twenty times as great in acid whey as in sweet whey.

Mineral content of dairy products. II. Cheeses.

Twenty-one kinds of commercial cheese were analyzed for fat, solids, protein, and five major and four trace minerals. Calcium, magnesium, sodium, and potassium were determined by atomic absorption; phosphorus was determined colorimetrically. Flameless atomic absorption was used to determine iron, zinc, copper, and manganese. The data agreed in general with published values, although there were instances of wide variation. The data represent a comprehensive compilation of the mineral contents of popular cheeses that should be helpful to dietitians and nutritionists.

Mineral content of dairy products. I. Milk and milk products.

Sixteen kinds of dairy products were analyzed for five major minerals and four trace minerals. Commercial samples of fluid milk, cream, concentrated milks, cultured products, butter, and frozen desserts were also analyzed for fat, solids, protein, and minerals. Calcium, magnesium, sodium, and potassium were determined by atomic absorption, and phosphorus was determined colorimetrically. Flameless atomic absorption was used to determine iron, zinc, copper, and manganese. The data generally agreed with most recently published values. However, for the trace minerals, the data differed widely in some instances. Manufacturing practices and added ingredients produced considerable variations in mineral content of market samples. These variations, however, could be limited by selection of products, so that they would not preclude the use of dairy products in diets in which mineral composition must be controlled. The coefficients of variation, which indicate the variability that can be expected for each product, generally were high for sherbet and ice milk and low for low-fat milk and skim milk.

Trace minerals in cottage cheese.

The trace minerals, iron, zinc, copper, and manganese were determined in cottage cheese curd by flameless atomic absorption. Larger curd size, added calcium chloride, and fewer washes increased the iron and zinc content in the curd.

Composition of cheddar cheese made with different milk clotting enzymes.

Six commercial milk clotting preparations from animal and fungal sources were used to make cheddar cheese. The cheeses were analyzed initially and over 6-mo ripening for proximate composition, minerals, amino acids, soluble protein, nonprotein nitrogen, free fatty acids, lactones, and flavor development. No significant differences in the composition of the cheeses could be attributed to the type of clotting enzyme. One lot of one enzyme showed increased lipolytic activity which indicated contamination and suggested that the purity of the enzyme preparation should be checked.