Sample preparation and liquid chromatographic determination of vitamin D in food products

Vitamin D in different fortified foods is determined by using liquid chromatography (LC). Sample preparation is described for fortified skim milk, infant formulas, chocolate drink powder, and diet food. The procedure involves 2 main steps: saponification of the sample followed by extraction, and quantitation by LC analysis. Depending on the sample matrix, additional steps are necessary, i.e., enzymatic digestion for hydrolyzing the starch in the sample and cartridge purification before LC injection. An isocratic system consisting of 0.5% water in methanol (v/v) on two 5 microns ODS Hypersil, 12 X 0.4 cm id columns is used. Recovery of vitamin D added to unfortified skim milk is 98%. The results of vitamin D determination in homogenized skim milk, fortified milk powder, fortified milk powder with soybean, chocolate drink powder, and sports diet food are given.     

Liquid chromatographic determination of vitamin D in fortified milk

A method for the determination of vitamins D2 + D3 in fortified milk is described. Vitamins D2 and D3 are extracted from the saponified sample and converted to isotachysterols with antimony trichloride. The isotachysterols are quantitated using liquid chromatography with ultraviolet detection at 301 nm, which is the absorption maximum. At this wavelength other materials present in the sample do not interfere with the analysis of isotachysterols and therefore a cleanup step is avoided. Recoveries of vitamin D added to skim milk were 98.1% (SD 5.3), 96.7% (SD 3.3), and 96.0% (SD 5.1) for samples fortified with 200, 400, and 600 IU/quart, respectively. For whole milk, recoveries were 102.0% (SD 6.5) and 97.1% (SD 3.5) in samples fortified with vitamin D equivalent to 200 and 400 IU/quart, respectively. The detection limit for vitamin D is 40 IU/quart.     

High pressure liquid chromatographic determination of vitamin D3 in instant nonfat dried milk

Vitamin D3 was determined in commercially fortified instant nonfat dried milk by using normal phase high pressure liquid chromatography (HPLC). The sample was extracted with dichloromethane with sodium phosphate tribasic solution added. The sample was cleaned up by using a Sep-Pak silica cartridge and then a microparticulate column containing 10 micrometer Partisil-10 PAC packing material. The final analysis was performed by using a normal phase HPLC system with 10 micrometer LiChrosorb NH2 column. Recovery of vitamin D3 at levels as low as 10000 IU/kg was 97.7% with a standard deviation of 3.9%.     

Liquid chromatographic determination of vitamins D2 and D3 in fortified milk and infant formulas

A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Survey of vitamin content of fortified milk

This paper summarizes work done by 4 different laboratories on the vitamin content of milk. Riboflavin, vitamin A, and vitamin D were assayed in whole, 2%, and skim milks that had been fortified. In general, the adherence to label claim decreased with decreasing fat content. This may be due to methods and stage of vitamin addition prior to processing.     

Liquid chromatographic determination of vitamin D in milk and infant formula

Vitamin D2 or vitamin D3 is determined by liquid chromatography (LC) in milk and infant formula. Vitamin D is extracted from the saponified sample, passed through an amino-cyano LC cleanup column to remove major interferences, and quantitated using normal phase LC. Within-day precision is 4.5% relative standard deviation (RSD); the overall method RSD (reflecting technician-to-technician, day-to-day, and within-day variability) is 7.7%. Overspike recoveries averaged 97% for milk, 98% for milk-based infant formula, and 93% for soy-based infant formula. The performance of the method is compared with that of the official AOAC vitamin D method (rat bioassay). The method is applicable to the determination of vitamin D in milk and in the major milk- and soy-based infant formulas available in the United States. The method can quantitate (but not distinguish) either vitamin D2 or vitamin D3. The method is applicable to milk and infant formula samples containing between 100 and 1500 IU vitamin D/L. Sample throughput is between 4 and 8 replicates per day.     

Analysis of fat-soluble vitamins. XXIII. High performance liquid chromatographic assay for vitamin D in vitamin D3 and multivitamin preparations.

Vitamin D is determined in preparations containing other fat-soluble vitamins by high performance liquid chromatography (HPLC). The unsaponifiable residue is extracted and separated from interferences by reverse phase chromatography; the fraction corresponding to vitamin D3 is collected and quantitated using normal phase chromatography (amylalcohol-n-hexane as mobile phase) by measuring the vitamin D3 and pre-vitamin D3 peaks at 254 nm. Previtamin D3 content is calculated as vitamin D3 with a conversion factor (determined on the equipment used). Application of the method to vitamin AD3 mixtures in oils gives 98-102% recovery. The reproducibility, using an external standard, is 2-3%, calculated as the coefficient of variation; with an internal standard, the coefficient of variation is 1-1.5%. The method measures potential vitamin D3 content in preparations containing greater than or equal to 200 IU/g in the presence of all known vitamin D3 isomers, vitamin A, and vitamin E.     

Reverse phase high pressure liquid chromatography of vitamin A in margarine, infant formula, and fortified milk

Retinal was determined in margarine (50 mg), infant formula (1 ml), and fortified milk (1 ml) by saponification in centrifuge tubes, extraction of the unsaponifiable lipid with hexane, and high pressure liquid chromatography (HPLC) in 90% methanol on a 25 cm x 3.2 mm column containing 10 micrometer LiChrosorb reverse phase. beta-Carotene was determined using the same column and 99% methanol as eluant. The vitamins in the eluate were identified and measured from their absorption at 325 (retinol) and 453 nm (beta-carotene), using a computing integrator. Retinol and carotene were prominent peaks in the chromatograms from properly fortified samples and were satisfactorily separated from other materials. In a survey of 12 different margarines for retinol, the results from liquid chromatography agreed with those of a variety of spectrophotometric and fluorometric procedures but they were obtained with greater ease and they could be interpreted more confidently. The recovery of retinol from oil was better than 99%. The coefficients of variation was 6.8% for 12 replicate analyses of a margarine for retinol and 5.4% for 10 replicate analyses of a margarine for beta-carotene.     

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.     

Vitamin D3 fortification, quantification, and long-term stability in Cheddar and low-fat cheeses

Considering the widespread insufficiency of vitamin D, the fortification of additional foods with vitamin D is warranted. The objective of this research was to assess the feasibility of vitamin D3 fortification in natural hard cheeses. We examined the recovery, distribution, long-term retention, and heat stability of the vitamin in industrially made fortified Cheddar and low-fat cheeses. The results indicated that the vitamin D3 did not degrade during processing, over 1 year of ripening (3-8 degrees C), or after thermal treatment at 232 degrees C for 5 min. Vitamin D3 recovery in the fortified Cheddar and low-fat cheeses were, respectively, 91 and 55% of the vitamin D3 added to the milk used to make each cheese. The remaining vitamin D3 was entrained in the whey. The vitamin D3 was uniformly distributed throughout the blocks of cheese. The fortification process did not alter the yield, chemical composition, or flavor of the Cheddar cheese. We conclude that industrially manufactured Cheddar and low-fat cheeses are suitable for vitamin D3 fortification.     

Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.     

High pressure liquid chromatographic separation and identification of vitamins D2 and D3 in the presence of fat-soluble vitamins in dosage forms.

A simple and rapid qualitative method is described for determining the presence of vitamin D2 (ergocalciferol) and/or vitamin D3 (cholecalciferol) in various preparations by reverse phase high pressure liquid chromatography (HPLC). When both D2 and D3 are present, this method effectively separates and identifies each vitamin D form by its respective retention time. A significant difference between vitamins D2 and D3 exists in their antirachitic activity in poultry. Preparations can be tested rapidly by this method to ascertain that the correct D vitamin form has been added. Fat-soluble vitamins such as vitamins A, E, K1, and K3 do not interfere. Vitamins D2 and D3 were separated at the baseline in model preparations. As little as 2 ng each of vitamin D2 and vitamin D3 can be separated and identified.     

Antiosteoporotic effects of Lactobacillus -fermented soy skim milk on bone mineral density and the microstructure of femoral bone in ovariectomized mice

Osteoporosis is a major skeletal disease associated with loss of estrogen in postmenopausal women. In this study, Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101F) and Lactobacillus plantarum NTU 102 (NTU 102F) were used as starters to ferment soy skim milk. This was then used as a nutritional supplement for 8 weeks to ovariectomized (OVX) mice. This study reveals that soy skim milk fermented with lactobacilli can increase the contents of aglycone isoflavones, soluble calcium, and vitamin D(3). The trabecular bone volumes and trabecular number of the distal femur in mice fed NTU 101F increased by a factor of 1.48 and 1.74 compared with the OVX group. The bone network density and thickness of the distal metaphyseal trabecular in mice fed NTU 101F and Fosamax was significantly greater than that of OVX mice. These results suggest that fermented soy skim milk can attenuate bone loss in OVX mice and lower the risk of osteoporosis.     

Modification of milk and whey surface properties by enzymatic hydrolysis of milk phospholipids

Phospholipase A1 were shown to improve foaming properties of skim milk and whey, implying that phospholipases can be useful tools for modifying the functionality of dairy products and ingredients. The ability of three fungal phospholipases and porcine pancreatic phospholipase A2 to hydrolyze milk phospholipids was investigated by using sodium deoxycholate-solubilized milk phospholipid and whole milk. The enzyme with the highest activity in milk was Fusarium venenatum phospholipase A1. Milk and whey were subsequently characterized using tensiometry and interfacial shear rheology. The lysophospholipids released from the fat globule membrane decreased the surface tension of skim milk and whey. A dramatic decrease in the surface shear viscous and elastic moduli indicated a shift from a protein-dominated to a surfactant-dominated interface. The surface shear moduli did not correlate with the foam stability, which was improved by phospholipase A1.     

Automated fluorometric determination of vitamin A in milk

A previously published fluorometric method for vitamin A in milk, involving saponification in centrifuge tubes and extraction with hexane, was automated using conventional automated equipment including a filter fluorometer. The extraction and separation steps were affected by the presence of fat, so standards were prepared in milk. The peaks obtained with samples were 95% of steady state values. The coefficients of variation for 10 replicate analyses were 1.9% for unfortified milk and 1.3% for fortified milk. In tests on 19 different milk samples, the results of the automated and manual fluorometric methods differed by less than 10%; a colorimetric method using SbCl3 gave more erratic results and was more lengthy and laborious.     

Comparison of Carr-Price analysis and liquid chromatographic analysis for vitamin A in fortified milk

The determination of the vitamin A concentration in fortified milk was compared using Carr-Price analysis and liquid chromatography (LC). Carr-Price analysis required saponification of the sample with alcoholic potassium hydroxide, extraction with ether, and colorimetry with antimony trichloride in chloroform. LC analysis required hexane extraction of a 71% alcohol-sample solution and centrifugation at 2000 rpm. A 100 microL aliquot of the extract was analyzed on a LiChrosorb Si-60, 5 micron column, using an ethyl ether-hexane (2 + 98) mobile phase and detection at 313 nm. Each method was statistically evaluated for precision and sample-to-sample reproducibility. The LC extraction procedure was examined for efficiency. Each LC value was divided by the Carr-Price value obtained for the same sample; an average value of 0.975 with a coefficient of variation of 6.90% was obtained. It was concluded that the procedures were statistically equivalent.     

High pressure liquid chromatographic determination of nitrofurazone in milk.

A rapid high pressure liquid chromatographic (HPLC) screening method for the quantitative determination of nitrofurazone in milk has been developed. The drug is extracted with ethyl acetate from a 2.0 ml milk serum sample, the organic layer is evaporated to dryness, and the residue is dissolved in the mobile phase and injected into the liquid chromatogarph. A reverse phase muBondapak C18 column is used with monitoring at 365 nm. The detection limit is 5 ppb and recoveries are 57--67%. Mass spectroscopic confirmation of the HPLC nitrofuran peak is described.     

Quantification of milk fat in chocolate fats by triacylglycerol analysis using gas-liquid chromatography

The development and in-house testing of a method for the quantification of milk fat in chocolate fats is described. A database consisting of the triacylglycerol profiles of 310 genuine milk fat samples from 21 European countries and 947 mixtures thereof with chocolate fats was created under a strict quality control scheme using 26 triacylglycerol reference standards for calibration purposes. Out of the individual triacylglycerol fractions obtained, 1-palmitoyl-2-stearoyl-3-butyroyl-glycerol (PSB) was selected as suitable marker compound for the determination of the proportion of milk fat in chocolate fats. By using PSB values from the standardized database, a calibration function using simple linear regression analysis was calculated to be used for future estimations of the milk fat content. A comparison with the widely used butyric acid method, which is currently used to determine the milk fat content in nonmilk fat mixtures, showed that both methods were equivalent in terms of accuracy. The advantage of the presented approach is that for further applications, i.e., determination of foreign fats in chocolate fats, just a single analysis is necessary, whereas for the same purpose, the C4 method requires two different analytical methods.     

Determination of cocoa butter equivalents in milk chocolate by triacylglycerol profiling

An analytical approach for the detection and quantification of cocoa butter equivalents (CBEs) in milk chocolate is presented. It is based on (i) a comprehensive standardized database covering the triacylglycerol composition of a wide range of authentic milk fat (n=310), cocoa butter (n=75), and CBE (n=74) samples and 947 gravimetrically prepared mixtures thereof, (ii) the availability of a certified cocoa butter reference material (IRMM-801) for calibration, (iii) an evaluation algorithm, which allows a reliable quantification of the milk fat content in chocolate fats using a simple linear regression model, (iv) a subsequent correction of triacylglycerols deriving from milk fat, (v) mathematical expressions to detect the presence of CBEs in milk chocolate, and (vi) a multivariate statistical formula to quantify the amount of CBEs in milk chocolate. The detection limit was 1% CBE in chocolate fat (0.3% CBE in milk chocolate, having a fat content of 30%). For quantification, the average error for prediction was 1.2% CBE in chocolate fat, corresponding to 0.4% in milk chocolate (fat content, 30%).     

Determination of conjugated linoleic acid (CLA) concentrations in milk chocolate

The fatty acids from a series of milk-chocolate-based confectionery samples were analyzed as methyl esters by GC to determine the presence and amount of conjugated linoleic acid (CLA). A single peak corresponding to the 9-cis,11-trans isomer and ranging from less than 0.1% to nearly 0.2% of the total fatty acids, corresponding to up to 0.3 mg per g of chocolate, was observed. One of the chocolate extracts and a milk extract were subjected to silver ion HPLC and GC-MS in order to confirm the identity of the major isomer and tentatively identity minor isomers.