Single laboratory method performance evaluation for the analysis of total food folate by trienzyme extraction and microplate assay.

Single laboratory method performance parameters, including the calibration curve, accuracy, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ), were evaluated for the analysis of total food folate by the trienzyme extraction and microplate assay with Lactobacillus casei subsp. rhamnosus. Standard Reference Material (SRM) 1546 (meat homogenate), SRM 2383 (baby food composite), SRM 1846 (infant formula), Certified Reference Material (CRM) 121 (wholemeal flour), and CRM 485 (mixed vegetables), representing a broad selection of food matrices, were used to evaluate the performance of the method. A generated 4-parameter logistic equation of the calibration curve was y= (0.0705 - 1.0396)/(1 + (x/0.0165) (1.3072)) + 1.0396 (P < 0.0001). The test of parallelism demonstrated that matrix components in the food extracts did not affect the accuracy. Measured values of the SRMs and CRMs were within their certified or reference values. Recoveries for all reference materials met the requirements of the AOAC guidelines for single laboratory validation. Precision measured as repeatability, including simultaneous and consecutive replicates for each SRM and CRM, met the Horwitz criterion. LOD and LOQ values were 0.3 and 0.6 mug/100 g, respectively. The results showed that trienzyme digestion using alpha-amylase, Pronase(R), and conjugase from chicken pancreas coupled with a 96-well microplate assay provided a highly accurate, reproducible, and sensitive method for the determination of folate in a variety of foods.

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula.

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.

The performance of broiler chicks fed diets containing extruded cottonseed meal supplemented with lysine.

Three experiments were conducted to test the hypothesis that extruding cottonseed meal (CSM) with supplemental lysine improves its feeding value by detoxifying gossypol. The performance of 1-wk-old straight-run Peterson x Arbor Acres broiler chicks fed diets containing 20% feed-grade or extruded CSM was compared with that of control chicks fed corn and soybean meal-based broiler rations. All diets were formulated to meet minimum NRC requirements. Lysine levels were adjusted by addition of synthetic lysine at rates of 0.5 to 2.0% of the protein in CSM. In all experiments, weight gain, feed intake, and feed conversion ratio (FCR) of broilers at 21 d were significantly affected by the diets. Feeding feed-grade and extruded CSM resulted in decreased body weight gain, increased feed intake, and inefficient feed utilization. When 2% lysine was added to feed-grade or extruded CSM, the body weight gains of chicks were not significantly different from those fed the control diet. The FCR of chicks fed feed-grade and extruded CSM plus 2% lysine at 21 d was significantly better than that of chicks fed feed-grade or extruded CSM alone. Abdominal fat pads (as a percentage of body weight) were significantly increased by the inclusion of CSM with or without the addition of lysine (P < or = 0.019). Liver, spleen, and heart weights were not affected by the presence of 20% CSM in the diet. The effects of CSM on plasma iron level was not consistent. Only in Experiment 1 did CSM cause a significant reduction in plasma iron. The hemoglobin contents and hematocrit values of blood from chicks fed diets with 20% CSM were not significantly different from those of the controls. The extrusion process reduced the free gossypol in CSM, but the total gossypol level was not changed, and chick performance was not improved. However, this study shows that, with adequate supplemental lysine, CSM can be used in broiler diets without a reduction in performance.

Comparison of the column performance of narrow-bore and standard-bore columns for the chromatographic determination of alpha-, beta-, gamma-, and delta-tocopherol.

A comparison of the performance of narrow-bore (2.1-mm i.d.) and standard-bore (4.6-mm i.d.) analytical silica columns having the same length is completed for the resolution of alpha-, beta-, gamma-, and delta-tocopherol. The studies are performed on high-performance liquid chromatographic equipment with minimum extracolumn contribution. Column permeabilities are 1.16 x 10(-9) and 2.48 x 10(-9) cm2 for narrow and standard bore, respectively. The narrow-bore column gives up to a 7 times increase in sensitivity compared with a standard-bore column at equivalent running times for the analytes. Approximately one-third solvent savings can be achieved with the narrow-bore column. Theoretical plates of the standard-bore column are higher than that of the narrow-bore column.

Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk.

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha-ergocryptine, and ergocristine are extracted from wheat with methanol-0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 microg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovine and 20-400 ng/g for ergotamine, ergocornine, alpha-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of <5 and <20 ng/g, respectively, for each ergot alkaloid tested.

Determination of vitamin K1 in medical foods by liquid chromatography with postcolumn reduction and fluorometric detection.

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and alpha-amylase and extracted with 1% sodium bicarbonate solution-isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 sigma), and the limit of quantitation is 27 pg (10 sigma) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r= 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 +/- 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 +/- 0.088 mg vitamin K/kg (K or K1?) (n = 31) with a coefficient of variation of 9.26.

Liquid chromatographic analysis of all-trans-retinyl palmitate, beta-carotene, and vitamin E in fortified foods and the extraction of encapsulated and nonencapsulated retinyl palmitate.

A liquid chromatographic method is described for the analysis of natural vitamin E homologues, all-rac-alpha-tocopheryl acetate, retinyl palmitate (encapsulated and nonencapsulated), and beta-carotene in various fortified foods. The vitamins are extracted in 2-propanol and hexane without saponification and quantitated by normal phase chromatography with fluorescence and visible detection. The sample components were identified using an on-line three-dimensional photodiode array detector, which permitted profiling of the 190-800 nm absorption spectrum of any chromatographic peak. The method showed linearity for the analytes in their respective calibration ranges. The percent recoveries for retinyl palmitate using starch- and gelatin-encapsulated standards were 101.0 +/- 1.0 and 100.1 +/- 0.9, respectively. The method measures six or more analytes in a single injection and differentiates between natural and synthetic forms of vitamin E.

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.

Liquid chromatographic analysis of vitamin K1 in milk-based infant formula with matrix solid-phase dispersion.

A liquid chromatographic method for vitamin K1 in milk-based infant formula is described. The vitamins are extracted from infant formula by matrix solid-phase dispersion and quantitated by reversed-phase chromatography with fluorescence detection. Vitamin K1 is converted to the fluorescent hydroquinone with a postcolumn zinc reductive reactor. The limit of detection is 12 pg, and the limit of quantitation is 38 pg on-column. Linear responses were obtained in the range 0.55-22.1 ng/ml (r2 = 0.9998). Recoveries of vitamin K1 from an analyte-fortified blank material for milk-based infant formula averaged 91.7% (n = 25). The method provides a rapid, specific, and easily controlled assay for vitamin K1 in fortified infant formula.

Analysis of beta-carotene in medical food by liquid chromatography with matrix solid-phase dispersion.

A liquid chromatographic method is described for analysis of beta-carotene in medical food. The nutrient is extracted from medical food without saponification by matrix solid-phase dispersion and quantitated by isocratic normal-phase chromatography with a Si 60 column and a mobile phase of hexane containing 0.125% (v/v) isopropyl alcohol. The limit of quantitation is 0.02 microgram/mL at 436 nm. Standard response was linear over the concentration range of 0.02-1.0 microgram/mL (r2 = 0.99998). Recoveries were determined on a zero control reference material containing added beta-carotene at various levels. Recoveries averaged 91.2% (n = 25) with coefficients of variation from 0.50 to 3.10%. The method provides a rapid, specific, sensitive, and easily controlled assay for analysis of beta-carotene in fortified medical food. In addition, retinyl palmitate can be assayed simultaneously with an in-line fluorescence detector.

Analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in medical foods using a zero control reference material (ZRM) as a method development tool.

A liquid chromatographic method is described for analysis of all- rac-alpha-tocopheryl acetate and retinyl palmitate in medical food. The vitamins are extracted in isopropyl alcohol and hexane-ethyl acetate without saponification and quantitated by normal-phase chromatography with fluorescence detection. All rac-alpha-tocopheryl acetate and retinyl palmitate are chromatographed isocratically with a mobile phase of 0.5% (v/v) and 0.125% (v/v) isopropyl alcohol in hexane, respectively. Recovery studies performed on a medical food zero control reference material (ZRM) fortified with the analytes averaged 99.7% (n = 25) for retinyl palmitate and 101% (n = 25) for all- rac-alpha-tocopheryl acetate. Coefficients of variation were 0.87-2.63% for retinyl palmitate and 1.42-3.20% for all-rac-alpha-tocopheryl acetate. The method provides a rapid, specific, and easily controlled assay for analysis of vitamin A and vitamin E in medical foods. Use of chlorinated solvents is avoided.

A liquid chromatographic method for analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in medical food using matrix solid-phase dispersion in conjunction with a zero reference material as a method development tool.

A liquid chromatographic method is described for analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in medical food. The vitamins are extracted from medical food without saponification by matrix solid-phase dispersion and chromatographed by normal-phase chromatography with fluorescence detection. Retinyl palmitate and all-rac-alpha-tocopheryl acetate are quantitated isocratically with a mobile phase of 0.125% (v/v) and 0.5% (v/v) isopropyl alcohol in hexane, respectively. Results compared favorably with label declarations on retail medical foods. Recoveries determined on an analyte-fortified zero reference material for a milk-based medical food averaged 98.3% (n = 25) for retinyl palmitate spikes and 95.7% (n = 25) for all-rac-alpha-tocopheryl acetate spikes. Five concentrations were examined for each analyte, and results were linear (r2 = 0.995 for retinyl palmitate and 0.9998 for all-rac-alpha-tocopheryl acetate) over the concentration range examined, with coefficients of variation in the range 0.81-4.22%. The method provides a rapid, specific, and easily controlled assay for analysis of retinyl palmitate and all-rac-alpha-tocopheryl acetate in fortified medical foods.

High dietary iron enhances oxidative stress in liver but does not increase aberrant crypt foci development in rats with low vitamin E status.

The purpose of this study was to examine the effects of high-iron and low-vitamin E diets on lipid peroxidation and aberrant crypt foci (ACF) development in rats. In a 2 x 2 x 2 factorial design, male Sprague-Dawley rats were fed 45 or 450 mg Fe/kg diet (adequate and high iron, respectively) and 15 or 100 IU vitamin E/kg diet (low and adequate vitamin E, respectively) for three weeks, when they received saline or azoxymethane (15 mg/kg for 2 wk). Diets were continued for an additional six weeks. Serum alpha-tocopherol concentrations in rats fed low-vitamin E diets were decreased to 30% of concentrations observed in rats fed adequate-vitamin E diets (p < 0.0001). Also, serum alpha-tocopherol concentrations tended to be lower in rats supplemented with iron (p < 0.08). Lipid peroxidation in liver was significantly elevated by high-iron diets after 3 and 10 weeks of treatment, but lipid peroxidation in colonic mucosa was not altered by dietary iron or vitamin E. The total number of ACF and number of large ACF (> or = 4 aberrant crypts/focus) were not significantly altered by iron or vitamin E intakes. However, the size distribution of ACF was slightly altered, such that iron-supplemented rats had 12% more ACF with two crypts per focus (p < 0.02) than rats fed adequate-iron diets. Our data suggest that high-iron diets enhanced oxidative stress in liver, but not colon, of rats fed low-vitamin E diets. Furthermore, a high-iron diet does not increase the total number of ACF, even when vitamin E status is low.

Liquid chromatographic method for analysis of All-rac-alpha-tocopheryl acetate and retinyl palmitate in soy-based infant formula using a zero-control reference material (ZRM) as a method development tool.

A liquid chromatographic method is described for analysis of all-rac-alpha-tocopheryl acetate, tocopherols, and retinyl palmitate in soy-based infant formula. The vitamins are extracted in isopropyl alcohol and hexane--ethyl acetate without saponification and quantitated by normal-phase chromatography with fluorescence detection. All-rac-alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with mobile phases of 0.5% (v/v) and 0.125% (v/v) isopropyl alcohol in hexane, respectively. Recoveries from zero control reference material soy-based formula averaged 97.2% (n = 25) for retinyl palmitate and 100% (n = 25) for all-rac-alpha-tocopheryl acetate. Coefficients of variation ranged from 1.21 to 2.86% for retinyl palmitate and from 1.49 to 5.16% for all-rac-alpha-tocopheryl acetate. The method provides a rapid, specific, and easily controlled assay for analysis of vitamin A and vitamin E in fortified infant formula. Additionally, the method eliminates use of chlorinated solvents.

Zero control reference materials for infant formula methods development.

A zero control reference material (ZRM) for milk and soy-based infant formula was manufactured and characterized. The ZRM was free of retinyl palmitate and all-rac-alpha-tocopheryl acetate. The composition was similar to commercially available infant formula. The ZRM provides a valuable tool to ascertain method performance.

The folic acid requirements of starting broiler chicks fed diets based on practical ingredients. 1. Interrelationships with dietary choline.

Five experiments were conducted to evaluate the effect of dietary supplemental folic acid in starting broiler chick diets. In the first two experiments, basal diets based on corn and soybean meal contained 10 micrograms/kg vitamin B12 but no supplemental methionine or choline. Chicks showed curvilinear responses to folic acid supplementation with maximum growth and feed efficiencies from 1.45 mg/kg diet. The liver folic acid response was also curvilinear but reached a plateau at 1.70 mg folic acid/kg diet. The basal diet for three additional experiments contained soybean meal that had been washed with methanol to remove most of the choline. The basal diet contained only 750 mg/kg choline. Chicks exhibited a larger growth response to folic acid at low choline levels as evidenced by a significant folic acid by choline interaction. Choline and folic acid both increased tibia length and width. Folic acid supplementation increased but then decreased valgus deformity. Choline chloride supplementation also decreased the incidences of valgus and varus deformities and decreased bone ash, but increased the incidence of tibial dyschondroplasia. It is concluded that chicks fed practical ingredient-based diets require 1.3 mg folic acid/kg diet with low levels of choline, but only 1.2 mg folic acid/kg when choline is offered near the NRC recommended level of 1,300 mg/kg of choline.

The folic acid requirements of starting broiler chicks fed diets based on practical ingredients. 2. Interrelationships with dietary methionine.

Two experiments were conducted to determine the effects of dietary supplemental folic acid and methionine on the performance of starting broiler chicks for 18 d. Four levels of dietary folic acid (.24, .54, 1.14, and 2.34 mg/kg) and four levels of dietary methionine (.45, .53, .61, and .69%) were fed in a factorial design. There were three replicates of eight chicks each per each treatment. The basal diet was based on corn, isolated soybean protein, meat and bone meal, and fish meal. It contained adequate amounts of all nutrients except methionine and folic acid. Increased growth was observed in chicks fed the basal diet supplemented with either folic acid or methionine. Total dietary folic acid and methionine plus cysteine requirements for maximum growth were estimated to be 1.80 mg/kg and .85% in Experiment 1 and 1.47 mg/kg and .87% in Experiment 2, respectively. There were interactions between dietary folic acid and methionine on weight gain in both experiments. Chicks fed the diet containing 2.34 mg folic acid/kg tended to have depressed growth, as in previous experiments. There was a significant linear feed conversion response to folic acid in Experiment 1 and to methionine in Experiment 2. There were both linear and quadratic liver folic acid responses to dietary folic acid in both experiments. There was no indication that dietary methionine had any effect on liver folic acid content. No differences in bone ash, hemoglobin, hematocrit, or incidence of tibial dyschondroplasia were detected due to methionine or folic acid supplementation.

Method modification for liquid chromatographic determination of thiamine, riboflavin, and pyridoxine in medical foods.

A reversed-phased ion pair liquid chromatographic method developed for the simultaneous determination of thiamine (B1), riboflavin (B2), and pyridoxine (B6) in perchloric acid extracts of infant formulas was modified to include medical foods. UV detection of B1 and B2 was replaced by fluorescence detection, which resulted in improved sensitivity and specificity. B1 was detected by fluorescence after conversion to thiochrome by a postcolumn reaction with sodium hydroxide and potassium ferricyanide. The method uses a mobile phase of water, acetonitrile, hexanesulfonic acid sodium salt, ammonium hydroxide, and phosphoric acid adjusted to pH 3.6. The column is a 300 x 3.9 mm Nova Pak C18. Limits of detection were 0.05 microgram/mL for B1 and B2 and 0.01 microgram/mL for B6 by fluorescence detection. The system reproducibility was evaluated by completing 10 repetitive determinations on a medical food that gave a coefficient of variation of 5.9, 6.0, and 10.7% for B1, B2, and B6, respectively. Mean recoveries (n = 10) were 111, 96.3, and 113% for B1, B2, and B6, respectively. The results compared favorably with those by AOAC Official Methods 942.23, 940.33, and 961.15 for B1, B2, and B6, respectively.

Liquid chromatographic analysis of niacin in fortified food products.

An ion exchange liquid chromatographic (LC) method using an anion exchange resin column was developed for the determination of niacin in fortified foods. Samples were extracted by autoclaving with H2SO4 (1 + 1). Florisil open column chromatography was used to remove interferences from the sample extracts. Niacin levels were quantitated by an LC system using a 250 x 4.1 mm Hamilton PRP-X100 column, a mobile phase of 2% glacial acetic acid in water, and UV detection at 254 nm. The limit of detection was 0.11 micrograms niacin/mL, and the standard curve was linear from 0.24 to 0.80 micrograms niacin/mL. The system reproducibility was evaluated by completing 10 repetitive analyses on an infant formula and a macaroni product, which gave an average CV of 2.7%. Mean recovery (+/- standard deviation) was 99.8 +/- 7.7 (n = 15). The results compared favorably with those by the AOAC microbiological method.

Application of a tri-enzyme extraction for total folate determination in foods.

A tri-enzyme digestion procedure using chicken pancreas conjugase, alpha-amylase, and Pronase was evaluated to determine its usefulness in the microbiological quantitation of total folate in foods. Folate values obtained by traditional conjugase digestion were compared to those obtained by the tri-enzyme method for 12 food products that represent diverse matrixes. The tri-enzyme treatment increased measurable folate from most foods when compared to levels found after conjugase digestion. Largest increases were noted for tuna fish (51%) and yogurt (33%) after tri-enzyme digestion. For the 12 foods, a mean increase of 19% in measurable folate was obtained with tri-enzyme treatment. The study shows that traditional conjugase treatment does not completely free folate from complex food matrixes before microbiological analysis. Further, as other investigations have suggested, current accepted methods for folate analysis may be underestimating folate levels in foods.