Analysis of fat-soluble vitamins. XXII. High performance liquid chromatographic determination of vitamin D in concentrates. Collaborative study

A collaborative study was carried out which compared the official chemical method (43.B14-43.B24), the HPLC method according to Hofsass et al. including maleic anhydride treatment, and the HLPC procedure according to De Vries et al. for vitamin D concentrates. A total of 396 samples were distributed to 33 collaborators for analysis. Five laboratories performed both the chemical and the HPLC methods. Five laboratories performed the Hofsass method and 16 laboratories performed the De Vries method. The results for the chemical method agreed with the theoretical values for the samples, and the standard deviation was comparable to that obtained in previous AOAC collaborative studies. Collaborative results for the Hofsass method were low. In addition, incorrect use of a fixed conversion factor (1/0.586) and necessity of a double chromatogrpahic system on a non-treated and a treated vitamin D sample reduce the effectiveness of the method. There were no adverse reactions to the DE Vries HPLC method. It is recommended that the method be adopted official first action as an alternative procedure for determining vitamin D in concentrates, excluding powders containing irradiated 7-dehydrocholesterol.     

Analysis of fat-soluble vitamins. XX. Determination of vitamin D in multivitamin preparations. Second collaborative study.

The official first action method for determining vitamin D in multivitamin preparations was modified. The method was collaboratively studied by 7 laboratories, using 6 preparations in oil. The preparations consisted of vitamin D at various levels and at various ratios (in w/w) in vitamin A. Three samples contained cholecalciferol and 3 samples contained vitamin D3 from vitamin D3 resin. After outliers were eliminated by the Dixon test, data were analyzed and averages were compared with amounts of vitamin D known to be in each sample. For samples with vitamin D: vitamin A ratios of 1:0.5, 1:5, and 1:10, the mean vitamin D recoveries were 98.8, 94.6, and 90.7%, respectively. The method has been adopted as official final action.     

Determination of total sulfur in soil and plant samples using sodium bicarbonate/silver oxide,dry ashing and ion chromatography

Abstract

The determination of total sulfur (S) in soils and plant tissue samples can be accomplished using a combination of sodium bicarbonate/silver oxide, dry ashing and ion chromatography(IC). The proposed method offers high precision and acceptable accuracy for samples with more than 0.02% S. At the same time this procedure makes use of standard analytical equipment used in other phases of soil, water and plant tissue analysis. Soil sample sizes larger than 0.1g or high total S or barite (BaSO₄) may not have acceptable S recoveries due to incomplete S oxidation or dissolution of BaSO₄. In order to optimize recoveries of total S in these special soil samples, varying (decreasing) sample sizes is required.     

Effects of grinding and storage for one month on retention of vitamin A in premixes and mineral supplements

Tested standardized procedures for handling premixes and mineral supplements from time of sampling to time of analysis for vitamin A have not been developed, which could account for some unexplained inconsistent and low analytical results. Grinding premix samples and storing them in a freezer for one month had little effect on amount of vitamin A found, but there was a significant loss (about 10%) after storage for one month at room temperature. Results on replicated determinations of vitamin A in unground and ground mineral supplements and on effect of storage were somewhat more variable than for premixes, but only the loss (about 12%) during storage for one month at room temperature was significant.     

Novel low-density lipoprotein (LDL) oxidation model: antioxidant capacity for the inhibition of LDL oxidation

A novel model of peroxyl radical initiated low-density lipoprotein (LDL) oxidation (LDL oxidation model for antioxidant capacity, or LOMAC) was developed to assess the free radical scavenging capacity of antioxidants and the extracts of natural products. A water-soluble free radical initiator, 2,2'-azobis(amidinopropane) dihydrochloride, was used at physiological temperature (37 degrees C) to generate peroxyl radicals to catalyze lipid oxidation of LDL isolated from human plasma samples. Headspace hexanal, a major decomposition product of LDL oxidation, was measured by a headspace gas chromatograph as an indicator of antioxidant capacity of different concentrations of pure antioxidants (vitamins C and E) and the extracts of natural products (fresh apple phytochemical extracts). All vitamin C and E and apple extract concentrations tested resulted in increasing partial suppression and delay of LDL oxidation. On the basis of the median effective dose (EC(50)) calculated for each compound or extract tested, the LOMAC value of 100 g of apple against LDL oxidation was equivalent to 1470 mg of vitamin E or to 402 mg of vitamin C. This study shows that the LOMAC assay can be routinely used to analyze or screen antioxidants or phytochemical extracts against LDL oxidation to prevent cardiovascular disease. The food-specific LOMAC values will be very useful as a new alternative biomarker for future epidemiological studies of cardiovascular disease.     

High pressure liquid chromatographic determination of vitamin D3 in livestock feed supplements.

Vitamin D3 is determined in livestock feed supplements by high pressure liquid chromatography (HPLC). Extracts of the samples are quantitated using normal phase chromatography. If interfering co-extractives are present, an aliquot of the extract is injected on the normal phase column, and the fraction corresponding to vitamin D3 is collected. The vitamin fraction is then further cleaned up and separated from interferences by reverse phase chromatography, and quantitated by measuring the ultraviolet absorption at 254 and 280 nm. The method measures actual vitamin D3 content in the presence of pre-vitamin D, tachysterol, isotachysterol, and vitamin A.     

Evaluation of metal and microbial contamination in botanical supplements

The sale of botanical dietary supplements in the United States is on the rise. However, limited studies have been conducted on the safety of these supplements. There are reports on the presence of undesired metals in some of the botanical dietary supplements. In this study, echinacea, garlic, ginkgo, ginseng, grape seed extract, kava kava, saw palmetto, and St. John's wort supplements manufactured by Nature's Way, Meijer, GNC, Nutrilite, Solaray, Sundown and Natrol, have been analyzed for lead, mercury, cadmium, arsenic, uranium, chromium, vanadium, copper, zinc, molybdenum, palladium, tin, antimony, thallium, and tungsten using inductively coupled plasma mass spectrometry. All samples were devoid of mercury contamination. Results indicated that the botanical supplements analyzed did not contain unacceptable concentrations of these metals. These supplements were also evaluated for microbial contamination, and most samples analyzed showed the presence of bacteria or fungi or both. Microbes were not counted nor were microbial counts determined in these samples.     

Stability and Dietary Contribution of Vitamin E Added to Bread

Daily intake levels of vitamin E in the range of 200–800 IU are now recommended for its antioxidant effect. However, only vitamin E supplements or fortified foods may provide these high intake levels. As a fortified food, breads were prepared containing 200, 400, 800, or 1,600 IU of added vitamin E (dl‐α‐tocopheryl acetate) per pound loaf. These levels of fortification exerted no adverse effects on bread quality. However, only about two‐thirds of the added vitamin E was retained (recovered) in the breads, with retention values showing no further significant change during the seven‐day shelf‐life of the product. In fresh breads, vitamin E retention values were nearly the same (range 66.3–68.5%, average 67.2%) at all levels of vitamin E tested; this may hold true for levels not tested. Factoring in an average retention value of 67.2%, and actual potency (81.8%) of the vitamin E source used, a 50‐g serving of bread fortified with 1,600 IU of vitamin E per loaf would provide nearly one‐fourth of a suggested daily intake of 400 IU.     

Analysis of fat-soluble vitamins. XIX. Collaborative studies on the chemical assay for vitamin D concentrates

In 1971, a chemical method for the assay of vitamin D in concentrates containing only vitamin D was collaboratively studied by 14 laboratories, using 6 different samples from 2 European manufacturers. On the basis of these results, the laboratories were divided into 2 groups: 5 with significant laboratory biases of greater than or equal to 2%, and 9 laboratories with nonsignificant bias. The 9 laboratories were subdivided into 2 groups which differed significantly as to reproducibility within laboratories. The reproducibility between laboratories, expressed as a standard deviation in per cent with 95% confidence limits, was 1.2% (confidence range 0.6-7.3) and 4.7% (confidence range 2.4-29.3) for 3 and 6 laboratories, respectively. A second collaborative test was performed in 1974, using 12 vitamin D resin samples in oil from 3 United States manufacturers, to compare 2 chemical vitamin D assay methods (with and without maleic anhydride) and to compare results from the chemical and biological methods; 9 laboratories participated in the chemical method study and 3 in the rat bioassay study. The correlation of results of the chemical method including maleic anhydride treatment and the rat bioassays was satisfactory. The reproducibility of the chemical method was about the same as that in the first collaborative test.     

Proton magnetic resonance spectroscopic determination of disulfiram: collaborative study

A proton magnetic resonance spectroscopic method for determining disulfiram in the bulk drug product and in the formulated material was collaboratively studied. The method depends on the use of chloroform-d as a solvent and hexamethylcyclotrisiloxane as the internal standard. No interference from tablet excipients was observed. The method is rapid and specific. Eighteen laboratories analyzed duplicate samples of a bulk drug product, a 250 mg tablet composite, and a 500 mg tablet composite. The average per cent results and standard deviations were 99.7 +/- 1.4, 100.9 +/- 2.0, and 99.9 +/- 2.2, respectively.     

Gas-liquid chromatographic determination of diethylstilbestrol in molasses-based liquid feed supplements.

A gas-liquid chromatographic (GLC) method is described for the determination of diethylstilbestrol (DES) in molasses-based liquid feed supplements, using dienestrol diacetate as the internal standard. A sample equivalent to 110 mug DES was diluted, acidified with dilute sulfuric acid, extracted with chloroform, and subjected to basic sodium acetate cleanup. The bis-(trimethylsilyl) acetamide derivative was prepared and determined by GLC. Interfering peaks and/or low recoveries were found to be related to emulsions and the procedure incorporates centrifugation to minimize these.     

Lead, fluoride, and other elements in bonemeal supplements

The Pb, Cd, F, Al, Cr, Cu, Fe, Mn, Mo, Ni, Ti, and Zn content of 20 commercial bonemeal supplements was determined. Samples were mineralized with nitric and perchloric acids prior to determination of all elements except F, for which a diffusion method was used. Pb and Cd were determined by differential pulse anodic stripping voltammetry, F was measured using an ion selective electrode, and all other elements were determined by inductively coupled argon plasma spectroscopy. The mean recoveries of Pb and F were 97 and 99%, respectively. The concentration range of Pb was 1.5-8.7 microgram/g. Cd was quantitated in only one sample at a level of 2.5 microgram/g; all other samples were estimated to contain less than 0.05 microgram Cd/g. The concentration of F ranged from 261 to 921 microgram/g.     

Application of isotope dilution analysis for the evaluation of extraction conditions in the determination of total selenium and selenomethionine in yeast-based nutritional supplements

Isotope dilution analysis (IDA) has been used to quantify total selenium, total solubilized selenium, and the selenomethionine (SeMet) amount in yeast and yeast-based nutritional supplements after acid microwave digestion and different enzymatic extraction procedures. For this purpose, both a (77)Se-enriched SeMet spike, previously synthesized and characterized in our laboratory, and a (77)Se(VI) spike were used. In the analysis of the nutritional supplements, the SeMet spike was added to the sample and extracted under different conditions, and the (78)Se/(77)Se and (80)Se/(77)Se isotope ratios were measured as peak area ratios after high-performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The formation of SeH(+) and mass discrimination were corrected using a natural SeMet standard injected every three samples. Similarly, total solubilized selenium was measured in the extracts after enzymatic hydrolysis using the (77)Se-enriched SeMet as a spike by direct nebulization without a chromatographic separation. To establish a mass balance, total selenium was also determined by IDA-ICP-MS on the yeast tablets after microwave digestion using (77)Se(VI) as a spike. Results showed that all enzymatic procedures tested were able to solubilize total selenium quantitatively from the solid. However, the recovery for the species SeMet, the major selenium compound detected, was seriously affected by the enzymatic procedure employed and also by the matrix composition of the supplement evaluated. For the yeast sample, SeMet recovery increased from 68 to 76% by the combined use of driselase and protease. For the nutritional supplements, the two most effective procedures appeared to be protease and driselase/protease, with a SeMet recovery ranging from 49 to 63%, depending upon the supplement evaluated. In the case of in vitro gastrointestinal enzymolysis, the results obtained showed 26-37% SeMet recovery, while the rest of selenium was solubilized as other unknown compounds (probably Se-containing peptides).     

Analysis of fat-soluble vitamins. XXVIII. High performance liquid chromatographic determination of vitamin D in pet foods and feeds: collaborative study

A high performance liquid chromatographic (HPLC) method for vitamin D in pet foods and feeds at low concentrations (2-8 IU/g = 50-200 ppb) was studied collaboratively. The procedure consists of the following purification steps: saponification, extraction of the unsaponifiable fraction, chromatography on alumina, cleanup on reverse phase HPLC, and quantitation with straight phase HPLC. The original method, developed by Knapstein, was simplified by deleting the quantitative TLC step. Six coded samples were distributed to 31 laboratories, along with a known sample containing 15 IU/g to allow practice of the rather complicated procedure. Eighteen collaborators returned their results. Results for the spiked samples show good recovery. The estimates of repeatability and reproducibility are 0.96 and 2.2 IU/g for spiked samples and 1.5 and 3.1 IU/g for commercial samples, respectively, which are considered acceptable for these low concentrations. The method has been adopted official first action.     

Analysis of fat-soluble vitamins. XXIX. Liquid chromatographic determination of vitamin D in AD concentrates: collaborative study

A simplified liquid chromatographic (LC) method for determining vitamin D in vitamin AD concentrates (greater than or equal to 5000 IU vitamin D/g) was collaboratively studied. In the simplified method, the 2 columns specified in AOAC LC method 43.101-43.109 are replaced by a single column, which separates the vitamin D isomers and the vitamin A esters. The procedure for oils includes dissolution and quantitation by normal phase LC. Dry multivitamin concentrates and aqueous dispersions are treated with an enzyme system and the vitamins are extracted with n-pentane. Six coded samples were distributed to 16 laboratories; 15 collaborators returned their results. Estimates of repeatability and reproducibility for the oil samples were 1.1 and 3.1%, respectively; for the high-level concentrated dry preparation 1.4 and 3.9% and for the low-level concentrated dry preparation 1.3 and 11.4%, respectively. These values are a considerable improvement over the results obtained in the 1979 multivitamin collaborative study. The method has been adopted official first action for determination of vitamin D in vitamin AD concentrates containing greater than or equal to 5000 IU vitamin D/g.     

Collaborative study of a method for the detection of Clostridium botulinum and its toxins in foods.

The mouse toxicity and protection technique for the detection and identification of Clostridium botulinum and its toxins in foods was collaboratively studied by 11 laboratories. Each laboratory received 4 samples of cream of mushroom soup; 2 contained spores and toxin of C. botulinum type A, 1 contained spores and toxin of C. botulinum type E, and 1 contained spores of C. sporogenes. The media used were cooked meat medium (beef heart or chopped liver broth) and trypticase peptone glucose yeast extract broth with trypsin. The results indicate that this method has a high degree of repeatability and reproducibility. All 11 laboratories correctly identified the toxins and the nontoxic sample in the food and detected and identified the viable spores in the samples by means of the subsequent cultures. This method has been adopted as official first action.     

Gas-liquid chromatographic quantitation of phencyclidine.HCl in powders: collaborative study.

Six laboratories collaboratively studied a method for the quantitative gas-liquid chromatographic (GLC) determination of phencyclidine.HCl in powders. The phencyclidine.HCl and other water-soluble compounds are dissolved in dilute HCl. A portion of the aqueous solution is made weakly basic with K2HPO4, and the organic-soluble compounds are extracted in CHCl3 for the GLC determination of the phencyclidine.HCl. Eicosane (n-C20) is incorporated in the extracting CHCl3 as an internal standard. The samples collaboratively studied included samples of known phencyclidine.HCl concentration and one sample of unknown purity. Recoveries ranged from 92.1 to 104% and per cent standard deviations from 1.05 to 3.39. The method was adopted as official first action.     

Fecal coliform methods for examination of sea water: interlaboratory evaluation of split sample analysis.

An interlaboratory study was conducted to compare the effectiveness of the following 3 multiple-tube fermentation methods for determining the most probable number (MPN) of Escherichia coli in a split artificial sea water sample: (1) the 72-hr standard methods procedure of the American Public Health Association, (2) a 24-hr elevated-temperature test using A-1 medium, and (3) a 24-hr elevated temperature test modified to include an initial 3-hr resuscitation period using A-1 medium. The capability of the laboratories to perform the 3 test procedures was also compared. Split sample replicates with low, medium, and high levels of E. coli were examined in 18 laboratories in the United States and Canada. Data indicate that the laboratories performed each test with equal capability, and all 3 procedures were equally effective in enumerating the strain of E. coli used in this investigation. By virtue of its homogeneity and stability, the split sample served as an appropriate specimen for this study and could probably be used as a proficiency test specimen for evaluating laboratory analyst performance in the bacteriological examination of sea water.     

Enzymatic-ultraviolet method for measuring lactose in milk: collaborative study

Collaborators in 8 dairy and food industry laboratories performed one lactose determination on each of 8 unknown samples of milk, lowfat milk, or skim milk, as 3 pairs of blind duplicates. Two known samples were provided to gain experience prior to analysis of the unknown samples. All of the above samples were also analyzed for lactose content by the official AOAC gravimetric method (16.507) by a commercial laboratory. From the overall mean of results on all samples, determinations by the enzymatic method averaged 0.49% lower than by the AOAC method. This difference was significant by the t-test (P = 0.05), which indicated a lack of agreement between the compared methods in determining lactose content. Standard deviations were similar for the 3 sets of blind duplicates which ranged between 3.67 and 4.55% lactose content. F-values revealed that variations between means obtained by laboratories differed significantly as compared with variations within laboratory means. The method has been adopted official first action.     

Antioxidant activity of tocopherols, tocotrienols, and gamma-oryzanol components from rice bran against cholesterol oxidation accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride

The antioxidant activities of vitamin E (alpha-tocopherol, alpha-tocotrienol, gamma-tocopherol, and gamma-tocotrienol) and gamma-oryzanol components (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate) purified from rice bran were investigated in a cholesterol oxidation system accelerated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride. All components exhibited significant antioxidant activity in the inhibition of cholesterol oxidation. The highest antioxidant activity was found for 24-methylenecycloartanyl ferulate, and all three gamma-oryzanol components had activities higher than that of any of the four vitamin E components. Because the quantity of gamma-oryzanol is up to 10 times higher than that of vitamin E in rice bran, gamma-oryzanol may be a more important antioxidant of rice bran in the reduction of cholesterol oxidation than vitamin E, which has been considered to be the major antioxidant in rice bran. The antioxidant function of these components against cholesterol oxidation may contribute to the potential hypocholesterolemic property of rice bran.