calcium supplement

8/7/2019 calcium supplement

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United States Patent [19]Mazer et al.

I I I I I I ~ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ~ I I ~ I I I I I I I I I I I I I I IUS005698222A[11] Patent Number:[45] Date of Patent:

5,698,222Dec. 16, 1997

[54] CALCIUM SUPPLEMENT[75] Inventors: Terrence Bruce Mazer, Reynoldsburg;

NonnaneUa Torres DeWille,Columbus; Michael Allen Chandler;Robert John Ragan, both of Gahanna;Gregory Allan Snowden, Westerville;Maureen Elizabeth Geraghty,Columbus; Catherine DubininJohnson, Dublin; Lonnie RichardDrayer, Gahanna, all of Ohio

[73] Assignee: Abbott Laboratories, Abbott Park, Ill.[21] Appl. No.: 418,393[22] Filed: Apr. 7, 1995[51] Int. CI.6 A61K 9120[52] U.S. CI .......................... 424/464; 424/439; 424/500;

4241682; 514/904; 514/905[58] Field of Search 514/310,904,

514/905; 426n3; 4241439, 464, 500, 682[56] References Cited

U.S. PATENT DOCUMENTS3,949,0984;252,7974,737,3754,740,3804,786,5104,867,9894,956,1914,992;2825,158,9445;221,6685;260;2795,401,5245,438,0425,456,926

4/1976 Bangert 4261324211981 Rosenthal 514/524/1988 Nake1 et al 4261590411988 Me1achuris et al 42615901111988 Nake1 et al. 426n69/1989 Silva et al.....................42615911990 Ueda et al 4261330.3211991 Mehansho et al 426n2

10/1992 Makino et al 5141167611993 Henningfie1d et al 514/231111993 Greenberg 5141213/1995 Burkes et aI. .. 4261590811995 Schmid! et al 514121

10/1995 Hill et al 426n3FOREIGN PATENT DOCUMENTS

0486425 5/1992 European Pat. Off..0210024 11/1984 Japan.1118608 1/1968 United Kingdom.2196523 5/1988 United Kingdom.

9 11 19 69 2 1 211 99 1 WIPO.92/19251 1111992 WIPO.92/21355 1211992 WIPO.95/05808 3/1995 WIPO.

OTHER PUBUCATIONSWoodroof et al., Beverages: Carbonated and Noncarbon-ated, AVI Publishing, 1974, pp. 143-146.Melillo, Food Products Development, Jun. 1977, pp.108-110.Data From the National Health Survey, Series I T , No. 231,DHHS Pub. No. (PHS) 83-1681, p. 20 (1983).Melillo Physical Factors Governing the Stabilization ofCloudy Beverages, Jun., 1977, FoodProductDevelopment,pp. 108 to 110.Kelly et al., Gastroenterology, 87: 5%-600 (1984).Bei et al., American Journal of Clinical Nutrition,44:244-247 (1986).Beuchat, Food and Beverage Mycology, Van Nostrand Rein-hold, 1987, pp. 120-122.Smith et al., Calcified Tissue International, 41:351-352(1987).Nationwide Food Consumption Survey, USDA NFCS,Report 86-3, (1988), pp. 62 and 75.Spencer et al., Journal of Nutrition, 118:657-660, (1988).Champagne, Advances in Experimental Medicine and Biol-ogy, 249:173-184 (1989).Mehansho et al., Journal of the American College of Nutri-tion, 8(1):6H58 (1989).Churella et al., The FASEB Journal, 4(3):A788 (1990).Hanning et al., American Journal of Clinical Nutrition,54:903-908 (1991).

(List continued on next page.)Primary Examiner-Melvyn 1. MarquisAssistant Examiner-Robert H. HarrisonAttome): Agent, or Finn-Donald O. Nickey; Thomas D.Brainard; Lonnie R. Drayer[57] ABSTRACTA calcium supplement in solid form contains calciumglycerophosphate, vitamin D and vitamin C.

7 Claims, 11 Drawing Sheets

ETIfER/PENTANEEXTRACT

BROWN AQUEOUSLAYER


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5,698,222Page 2

OTHER PUBLICATIONSDraper et al., Journal of Parenteral and Enteral Nutrition,15(2):176-180 (1991).Sakhee et al., Bone and Mineral, 20:87-97 (1993).Wardlaw, Journal of the American Dietetic Association,93(9):1000-1006 (1993).Journal of the American Medical Association, 272(24):1942-1948 (1994).Whiting, Nutrition Reviews, 52(3):95-97 (1994).

56 FR 60689-60726 (1991).Label for Turns 500 TM.Label for Sunny Delight with Calcium.Label for Hawaiian Punch Double C .Federal Register, 58(3):2665-2681 (1993).Physicians DeskReference, 19th Edition, Published 1965, p.1015.Physicians DeskReference, 48th Edition, Published 1994, p.1155.


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u.s . Patent Dec. 16, 1997 Sheet 3 of 11 5,698,222

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u.s . Patent Dec. 16, 1997 Sheet 8 of 11 5,698,222CONSTANTCURRENTSOURCE

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u.s . Patent Dec. 16, 1997 Sheet 10 of 11 5,698,222

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u . s . Patent Dec. 16, 1997 Sheet 11 of 11

TIME

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LOC A TE ENDPOINT

FIG-12

5,698,222


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1CALC~SUPPLEMENTFIELD OF THE INVENTION

The present invention relates to a solid calcium supple- 5ment which is fortified with calcium glycerophosphate,vitamin D and vitamin C.

BACKGROUND OF THE INVENTION10Calcium is an essential nutrient; it is a major componentof mineralized tissues and is required for normal growth anddevelopment of the skeleton and teeth. Over the last decadecalcium has enjoyed increased attention due to its potentialrole in the prevention of osteoporosis. Osteoporosis affectsmore than 25 million people in the United States and is the 15

major underlying cause of bone fractures in postmenopausalwomen and the elderly. "Optimal Calcium Intake", JOUR-NAL OF THE AMERICAN MEDICAL ASSOCIATION,272(24): 1942-1948 (1994).As used herein "osteoporosis" refers to a reduction in the 20amount of bone mass. 1\\10 important factors influencing theoccurrence of osteoporosis are optimal peak bone massattained in the first two to three decades of life and the rateat which bone mass is lost in later years. Adequate calciumintake is critical to achieving optimal peak bone mass and 25modifies the rate of bone mass loss associated with aging.Wardlaw, ''Putting osteoporosis in perspective", JOURNALOF THE AMERICAN DIEfEI'IC ASSOCIATION, 93(9):1000--1006 (1993).Several cofactors modify calcium balance and influencebone mass. These include dietary constituents, hormones,drugs, and the level of physical activity. Unique host char-acteristics may also modify the effects of dietary calcium on

bone health. These include the individual's age and ethnicand genetic background, the presence of gastrointestinaldisorders such as malabsorption and the postgastrectomysyndrome, and the presence of liver and renal disease.Interactions among these diverse cofactors may affect cal-cium balance in either a positive or negative manner andthus alter the optimal levels of calcium intake. "OptimalCalcium Intake", JOURNAL OF THE AMERICAN MEDI-CAL ASSOCIATION, 272(24): 1942-1948 (1994).Calcium requirements vary throughout an individual'slifetime with greater needs occurring during the period ofrapid growth in childhood and adolescence, pregnancy andlactation, and in later adult life. Table 1 presents the optimalcalcium requirements which were established at a National

Institute of Health (NIH) conference on optimal calciumintake held Jun. 6-8, 1994. "Optimal Calcium Intake",JOURN AL OF THE AMERICAN MEDICALASSOCIATION,272(24): 1942-1948, at 1943 (1994). Theparticipants at the NIH conference considered former Rec-ommended Dietary Allowances (RDA) (10th edition, 1989)for calcium intake as reference levels and used them asguidelines to determine optimal calcium intake in light ofnew data on calcium-related disorders.TABLE 1

OPTIMAL CALCIUM INTAKES

GROUP

OPTIMAL DAILYINTAKE

(in ru g of calcium)InfantsBirth-6 months6 months-1 year

40060 0

5,698,2222

TABLE l-continuedOPTIMAL CALCIUM INTAKES

GROUPOPTIMAL DAILY

INTAKE(in ru g of calcium)Children1-5 years6-10 yearsAdolescentsiYOlmg Adults

800800-1,200

11-24 yearsMen

1,200-1,500

25-65 yearsOver 65 yearsWomen

1,0001,500

25-50 yearsOver 50 years (postmenopausal)

1,000

On estrogensNot on estrogensOver 65Pregnant and nursing

1,0001,5001,500

1,200-1,500

National consumption data indicate most females over theage of eleven, as well as elderly men, consume amounts ofcalcium below recommended levels. "Nationwide FoodConsumption Survey, Continuing Survey of Food Intakes of30 Individuals", USDA NFCS, CFS IT Report No. 86-93(1988), pages 62 and 75. According to the Second National

Health and Nutrition Examination Survey, the median dailycalcium intake for women in the United States was 574 mg.DIETARY INTAKE SOURCE DATA: UNIfED STATES,35 1976-80, Data From the National Health Survey, Series n,No. 231, DHHS Publication No. (PHS), 83-1681 (1983)page 20.The preferred approach to attaining optimal calcium

intake is through dietary sources. Dairy products are the40 major contributors of dietary calcium because of their high

calcium content (e.g. approximately 250--300 mg/8 oz ofcow's milk) and frequency of consumption. As used hereinthe term "milk' is understood to refer to cow's milk, and theterm "dairy products" is understood to refer to food products

45 derived from cow's milk. However, many persons, espe-cially women, prefer to limit their intake of dairy productsfor several reasons: (a) they dislike the taste of milk/milkproducts; and/or (b) they have a lactose intolerance; and/or(c) they perceive that some dairy products are too high in fat

50 or protein and may lead to weight gain. Other good foodsources of calcium include some green vegetables (e.g.broccoli, kale, turnip greens, Chinese cabbage), calcium-settofu, some legumes, canned fish, seeds and nuts. Breads andcereals, while relatively low in calcium, contribute signifi-

55 cantly to calcium intake because of their frequency ofconsumption. A number of calcium-fortified food productsare currently available, including fortified juices, fruitdrinks, breads and cereals. Consumption of these foods maybe an additional strategy by persons to achieve their optimal

60 calcium intake.To maximize calcium absorption, food selection decisions

should include consideration of information on the bioavail-ability of the calcium contained in the food. Bioavailability(absorption) of calcium from food depends on the food's

65 total calcium content and the presence of components whichenhance or inhibit calcium absorption. Bioavailability ofminerals in food has been traditionally tested by the balance


15/35

5,698,2223

method, which estimates absorption from the differencebetween ingested intake and fecal output. This approachworks well for many nutrients where the difference betweenintake and excretion is large, but is less well suited for anelement such as calcium entering the digestive tract with its 5secretions. A decline in fractional absorption from 30% to20% could have profound nutritional significance but wouldbe difficult to detect using the balance method. In contrast,isotopic methods estimate absorption directly from theappearance of the ingested tracer in body fluids. Future 10cliuical evaluations of the bioavailability of calcium fromthe liquid nutritional product of the present invention willuse a state-of-the-art isotope tracer method.Not all calcium salts are created equally. Calcium saltsrange from 9% elemental calcium in calcium gluconate to 15

40% calcium in calcium carbonate. Bioavailability dependson solubility. A new calcium delivery system, CalciumCitrate Malate (CCM) claims to be approximately six-timesthe solubility of either calcium citrate or calcium malate,both of which are themselves substantially more soluble 20than calcium carbonate. Smith et al., "Calcium Absorptionfrom a New Calcium Delivery System (CCM)" CALCI-FIED TISSUE INTERNATIONAL, 41: 351-352 (1987)relates an experiment in humans wherein calcium fromCCM was absorbed significantly better than from either 25calcium carbonate or milk. 38.3% vs 29.6% and 29.4%respectively. WO 91/19692 discloses a process for making ametastable calcium citrate malate.However, the United States Food and Drug Administra- 30

tion (FDA) has advised that, in order for calcium-containingfood ingredients in conventional foods or calcium supple-ment products to be considered eligible to bear the autho-rized calcium/osteoporosis health claim, they must meet therequirements in 101.14, which include that they have been 35shown to the FDA's satisfaction to be safe and lawful underthe applicable safety provisions of the act (56 FR at 60699).Safety and lawfulness can be demonstrated in a number ofways, including through a showing that a food is generallyrecognized as a safe (GRAS), affirmed as GRAS by the 40FDA, listed in the food additive regulations, or subject to aprior sanction. Of the 36 or more calcium-containing ingre-dients identified by the agency as currently in use the FDAadvised that only the following 10 compounds had beendemonstrated to be safe and lawful for use in a dietary 45supplement or as a nutrient supplement: calcium carbonate,calcium citrate, calcium glycerophosphate, calcium o~de,calcium pantothenate, calcium phosphate, calciumpyrophosphate, calcium chloride, calcium lactate, and cal-cium sulfate (56 FR at 60691).Table 2 summarizes the enhancement and inhibition fac-tors associated with calcium absorption.

TABLE 2FACIORS WInCH ENHANCE OR INHIBIT

CALCIUM ABSORPTIONInhibitors EnhancersOlder age (>51) YOImger age (11-24)Vitamin D deficiency Healthy vitamin D levelsOxalic acid, fiber & phytates (only Pregnancy & lactationifachlorhydria present)

Estrogen (natural & replacementtherapy)Caffeine Adequate protein intakePresence of other nutr ients in Ca+2 Ca+2:P04 ratio of 1:1supplement

4TABLE 2-continued

. FACTORS WInCH ENHANCE OR INHIBITCALCIUM ABSORPTION

Inhibitors EnhancersExcess protein intake > 2 X RDA Specific disaccharides: fructose &

lactoseSpecific organic acids:CitricMalicAscorbic

Calcium absorption is directly affected by an individual'svitamin D status. Vitamin D deficient individuals absorb lesscalcium than individuals whose vitamin D stores areadequate. Vitamin D metabolites enhance calcium absorp-tion. The major metabolite 1,25-dihydroxyvitamin D, stimu-lates active transport of calcium in the small intestine andcolon. Deficiency of 1,25-dihydroxyvitamin D, caused byinadequate dietary vitamin D, inadequate exposure tosunlight, impaired activation of vitamin D, or acquiredresistance to vitamin D, results in reduced calcium absorp-tion. In the absence of 1,25-dihydroxyvitamin D, less than10 percent of dietary calcium may be absorbed. Vitamin Ddeficiency is associated with an increased risk of fractures.Elderly patients are at particular risk for vitamin D defi-ciency because of insufficient vitamin D intake from theirdiet, impaired renal synthesis of 1,25-dihydroxyvitamin D,and inadequate sunlight exposure, which is normally themajor stimulus for endogenous vitamin D synthesis. This isespecially evident in homebound or institutionalized indi-viduals. Supplementation of vitamin D intake to provide600-800 IU/day has been shown to improve calcium bal-ance and reduce fracture risk in these individuals. Sufficientvitamin D intake should be ensured for all individuals,especially the elderly who are at greater risk for develop-ment of a deficiency. Sources of vitamin D, besides supple-ments include sunlight, vitamin D-fortified liquid dairyproducts, cod liver oil, and fatty fish. Calcium and vitaminD need not be taken together to be effective. Excessive dosesof vitamin D may introduce risks such as hypercalciuria andhypercalcemia and should be avoided. Anticonvulsant medi-cations may alter both vitamin D and bone mineral metabo-lism particularly in certain disorders, in the institutionalized,and in the elderly. Although symptomatic skeletal disease isuncommon in noninstitutionalized settings, optimal calciumintake is advised for persons using anticonvulsants. "Opti-50 mal Calcium Intake", JOURNAL OF THE AMERICANMEDICAL ASSOCIATION, 272(24): 1942-1948 (1994). Anumber of other dietary factors can also affect calciumabsorption. Dietary fiber and phytate have been implicatedas inhibiting substances. The binding of calcium by dietary

55 fiber increases with increasing pH. The onset of precipitationof calcium phytates occurs in the pH 4-6 range as inachlorhydria. At low gastric pH values (2-3), phytate doesnot bind calcium and calcium binding by dietary fiber wouldbe weak ifat all. Thus, in normal individuals calcium would

60 reach intestinal sites as soluble species. Depending on theconcentrations and binding strengths of various foodligands, some of the calcium will be absorbed at the intes-tinal sites while the remainder becomes bound as insolublefiber and phytate complexes. Champagne, "Low Gastric

65 Hydrochloric Acid Secretion and Mineral Bioavailability",ADVANCES IN EXPERIMENTAL MEDICINE ANDBIOLOGY,249: 173-184 (1989).


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5Simple sugars and organic acids also have an effect onbioavailability. Fructose in orange juice and apple juice

promoted positive calcium bioavailability from CalciumCitrate Malate (CCM) which is a combination of CaC03,citric acid, malic acid: 5:1:1 mol/mol/mol). The lactose inmilk forms a soluble compound with calcium. Organic acidssuch as citric acid, malic acid and ascorbic acid may alsoplay a role in the favorable absorption of calcium fromCCM. Mehansho et al., "Calcium Bioavailability and Iron-Calcium Interaction in Orange Juice", JOURNAL OF THEAMERICAN COLLEGE OF NUfRITION, 8(1): 61-68(1989).In addition, it is known that high protein intakes, specifi-cally of sulfur containing amino acids, increase urinarycalcium excretion. Sulfuric acid radicals are believed todecrease renal tubular resorption. However, consumption of

high phosphorus foods, such as meat, can diminish thiseffect Spencer et al., "Do Protein and Phosphorous CauseCalcium Loss?", JOURNAL OF NUfRITION, 118:657-660 (1988).For some individuals, calcium supplements may be thepreferred way to obtain optimal calcium intake. Althoughcalcium supplements are available in many salts, calciumcarbonate is usually recommended because it contains moreelemental calcium per gram than any of the other salts. Thedisintegration and dissolution characteristics of commercialcalcium carbonate preparations, which vary widely, mayproduce important differences in calcium absorption. Otherproblems with using large amounts of calcium carbonate isthat it can lead to constipation and abdominal distention.When problems arise, calcium lactate or calcium citrate are

advised. These substitutions for calcium carbonate are alsoindicated for people with achlorhydria. A popular commer-cially available calcium supplement is TUMS 500 whichis distributed by SmithKline Beecham, Pittsburgh, Pa.,U.S.A. and is labeled as providing 500 ru g of elemental 35calcium (from calcium carbonate per. tablet). However, theTUMS 500 label does not indicate that this calciumsupplement contains any vitamin D.U.S. Pat No. 4,786,510 and U.S. Pat. No. 4,992,282

disclose the use of calcium citrate malate in a beverage or 40dietary supplement fortified with iron, but do not disclosethe addition of vitamin D to such a product. WO 92119251and WO 92/21355 disclose the use of calcium citrate malatein a low pH beverage, and suggests that vitamin D be addedto such a beverage along with oil flavors or weighing oil. 45However; neither WO 92/19251 or WO 92-21355 discloseany other details about how to incorporate vitamin D3 intosuch a beverage.EP 0 486 425 A2 discloses a liquid oral nutritionalformulation which contains carbohydrates, protein, fat, fiber, 50calcium, and vitamin D, and has a pH of about 3.5 to 3.9.However, this publication teaches that high amounts ofrnicronutrients such as calcium or magnesium may impairthe palatability of the product, and should contain therecommended daily allowance of these nutrients in about 55one liter or product. In an example in the patent publicationthis product contains only about 570 mg of calcium per literand about 211 IU of vitamin D per liter. A commerciallyavailable product in accordance with this patent publicationis distributed by Sandoz Nutrition under the trade name 60CITRISOURCE and is labeled as providing 570 ru g ofcalcium and 210 IU of vitamin D per liter. By way ofcomparison, prototypes of a beverage according to thepresent invention contain about 1,408 rn g of calcium perliter and about 338 IU of vitamin D3 per liter.U.S. Pat. No. 4,737;375 teaches beverage concentrates

and beverages having a pH of 2.5 to 6.5, preferably 3.0 to

5,698,2226

4.5, which contains calcium. The use of vitamin D3 in thisbeverage is not disclosed. This patent does not teach the useof calcium glycerophosphate (which is used in preferredembodiments of the present invention, as a calcium source.5 The acidulants used in this prior art beverage are chosenfrom mixtures of citric acid, malic acid and phosphoric acid,and the weight ratio of total acids to calcium is in the rangeof 4 to 7. The calcium level is 0.06% to 0.15%, preferably0.10% to 0.15% of the beverage, by weight. By way of

10 comparison, prototypes of the beverage of the present inven-tionhave a weight ratio of total acids to calcium of about5.1.Two commercially available beverages which are labeled

as being protected by U.S. Pat. No. 7,737;375 are: (1) SunnyDelight With Calcium which is .distributed by Procter &Gamble, Cincinnati, Ohio 45202 U.S.A.; and (2) HAWAI-15 IANPUNCH, DOUBlE C which is distributed by SundorBrands, Inc., Cincinnati, Ohio 45202 U.S.A.. According tothe "Nutrition Facts" on the labels of these commerciallyavailable products: (a) either product contains vitamin D; (b)neither product contains any fat; (c) a 240 rnL (8 fluid ounce)

20 serving of Sunny Delight With Calcium provides 30% ofthe recommended daily intake of calcium; (d) a 240 rnL (8fluidounce) serving of HAWAIIANPUNCH, DOUBLE Cprovides 15% of the recommended daily intake of calcium;and (e) and a 240 rnL(8 fluid ounce) serving of each of these

25 products provides 100% of the recommended daily intake ofvitamin C. Per the product labels, these percent daily valuesare based on a 2,000 calorie diet. A review of the ingredientlistings on the labels of each of these products indicates thatboth of these beverages are aqueous solutions, and that

30 neither product contains gum arabic. Samples of each ofthese products were tested regarding their pH values: the pHvalue of the HAWAIIAN PUNCH DOUBlE C was 3.91;and the pH value of the Sunny Delight With Calcium was4.05.GB 2 1% 523 A discloses a beverage containing calcium

and vitamin D. A water soluble non-toxic calcium salt isused in a quantity sufficient to provide in the final beveragea calcium ion content of from 1.0xlO-2 to 40xl0-2% w/w.The beverage may contain up to 5xlO-6 wlw of vitamin D.However, this published patent application does not teachthe use of a gum, such as gum arabic or gum tragacanth, insuch a beverage to improve vitamin D3 stability.The NIH Consensus Statement recommended that theprivate sector play an active role in promoting optimal

calcium intake by developing and marketing a wide varietyof calcium-rich foods to meet the needs and tastes of amultiethnic population. "Optimal Calcium Intake", JOUR-NAL OF THE AMERICAN MEDICAL ASSOCIATION, 272(24): 1942-1948 (1994). Hence, there is provided in accor-dance with one aspect of the present invention a low pHbeverage fortified with calcium and vitamin D3. There isprovided in accordance with another aspect of the inventiona liquid beverage concentrate fortified with calcium andvitamin D3. There is provided in accordance with yetanother aspect of the invention a liquid beverage additivefortified with calcium and vitamin D3.

65

BRIEF DESCRIPTION OF THE DRAWINGSFIGS. 1-7 are representative of the methodology used in

determining vitamin D3 levels; andFIGS. 8-12 are representative of the methodology used in

determining vitamin C levels.DErAILED DESCRIPTION OF THE

lNVENTIONThe levels, half lives and other characteristics and prop-

erties of vitamin D3, calcium and vitamin C referred to


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26. SCOOp---VBteaspoon.B. HPLC Instrumentation1. Columns: Guard (4.6>99.7%)-for evaporations. 306. Vortex mixer--SIP Magnestir or equivalent.7. Volumetric flasks-lOO ml, 500 mi.8.Volumetric pipets-1 ml, 2 ml, 3ml, 5 ml, 7 ml, 15 ml,30 ml.

12. Therm-O-Vac-size 14120 (Cole-Parmer #N-06140-15).13. Teflon sleeves-sizes 24/40 (Cole-Parmer #N-06139- 5015).

14. Evapo-Rac Evaporator for 30 mm tubes (Cole-Parmer#N-01610-35).

15. Centrifuge tube rack (Cole-Parmer #N-06737-40).16. Cooling tray large enough to accommodate centrifuge 55tube rack (#N 06737-40).

17. HPLC tubing--O.040" stainless stee1-2 feet.18. Balances-(a) Merrier iU200 (or equivalent) readable to at least 600.01 mg (for standards, vitamin D3 emulsion andpowdered product.

(b) Mettler PM460 (or equivalent) readable to at least0.001 g (for low pH beverage samples).

19. Glass Stirring Rods.20. Magnestir Stir Plate-Lab Line #1250 or equivalent.21. Teflon Coated Stir Bars-2" length.

822. Beakers-600 ml, 800 ml, 1000 mi.23. Calculator-Hewlett Packard-llC or equivalent24. Refrigerator (freezer compartment optional) for stor-age of standards at 4(4)0 C.

25. Lighting Requirements Ultra-violet shields-F4aT12-Dayton Plastics Inc., for white fluorescentbulbs.

35

B. Chemicals

65

1. Amyl Alcohol Analyti ca l Reagent, recommend Mal linckrod tUN 1987.HPLC Grade, recommend Burdick & Jackson#230.HPLC Grade, recommend Burdick & Jackson#362.HPLC Grade, reconnnend Burdick & Jackson#312.

5. Diethyl Ether Anhydrous, reconnnend Mallinckrodt UN 11556. Potassium 45% solution, recommend Baker #3143-03.

2. Methanol3. Iso-octane4. Pentane


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5,698,2229

-continuedHydroxide7. Sodium RecommendAldrich #26,855-0.Ascorbate

8. Acetonitrile HPLC Grade, recommend Burdick & Jackson#015.9. Chlorofonn HPLC Grade, recommend Burdick & Jackson

#04810. Potassium Recommend Mallinckrodt #6838-500*NY.Chloride

11. n-Butyl HPLC Grade, recommend Burdick & JacksonChloride #034.

C. Solutions1. HPLC Mobile Phase Volumetrically pipet 40(O.I) ml 15of n-butyl chloride, 20(O.I) ml of amyl alcohol andlO(O.I) ml of chloroform into 4000 ml of iso-octane.Mix well. Make four liters at a time=-roughly equiva-lent to 1.0% n-butyl chloride +0.5% amyl alcohol+0.25% chloroform in iso-octane. Use for both cleanup 20and analytical HPLC systems. Completely fill the 10liter reservoir prior to each day's analysis.

2. Extraction Solutions#1-20:80 ether/pentane: Mix 200 ml of diethyl etherwith 800 ml of pentane. This is sufficient for up to 20 25samples (2)


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5,698,22211

12. Evaporate the transferred ether/pentane layer in thewarm water bath (4004 C.) with nitrogen to about 2m1 to allow for additional transfers. (See FIG. 2 for theEvapo-Rac evaporation apparatus.) 513. Repeat the extraction in steps #8-#12 once combiningthe extracts in the same 50 m1conical centrifuge tube.NOTE: Be careful not to overflow the 50 ml Corexcentrifuge tubes with the 25 m1extraction solutions (#1or #2) during the 2nd and 3rd extractions. Should thisoccur, the sample must be discarded and the assay 10repeated.

14. For the third extraction, follow steps #8-#12 using 25m1 of 33% ether/67% pentane solution (not the 20%ether/80% pentane solution)15. Evaporate the combined extractions to dryness.Remove the centrifuge tubes from the water bath assoon as evaporation is complete. The extract shouldappear clear or as a white or slightly yellow film. Makesure that the extract is completely dried before recon-stitution. The tubes may have to be gently tapped to 20complete the evaporation.

16. Immediately reconstitute with 2.0 (o.006) m1 ofiso-octane with a class A volumetric pipet Be carefulto thoroughly rinse down the walls of the tube. The tubeshould be tightly capped to prevent evaporation and 25vortexed 5 seconds to mix.17. Finally, add 1 ml of the KCL solution to each sampleand touch to the vortexer briefly to mix. Tightly cap andcentrifuge at moderate speed (approximately 300 X G)for 1 minute to complete phase separation. If using a 30centrifuge equipped with a swinging bucket rotor, placethe centrifuge tubes on the outside perimeter of therotor. This is to prevent the conical tubes from break-ing. Transfer only the top layer to a vial and tightly cap.Be careful not to transfer any of the saturated KCL 35solution. NOTE: The sample extract must be analyzedwithin 24 hours from time of preparation. Ifthe HPLCsystem encounters problems, the autos ampler vialshould be immediately stored below 8 C. after prepa-ration for up to 48 hours. No sample can be reinjected 40after an aborted HPLC analysis if it was left in theautosampler at room temperature overnight.

18. Inject onto the eqnilibrated HPLC system (section V).V. HPLC CONDmONSA. Cleanup HPLC System-See FIG. 3 for configuration. 45

1. Column: Chromegabond Cyano, (4.6 x 250 mID, 31l) with CS-GU guard column (4.6 x 30 mm).1.0% I-chlcrobutane + 0.5% amyl alcohol + 0.25%chloroform in iso-octane.Slice determination = approximately 20 minutes.1.5 mlImin.250!ll.4O(lt c.

2. Eluant:3. Run rune:4. Flow Rate:5. InjectionVolume:6. ColumnHeater:7. Detector:

8. Recorder:9. ColumnSwitch:

254 DIll or 264 nm,Integrator or data system (preferred).Actuated by timed control from injection point tocollection. Slice time window shou ld be no greaterthan 1.0 minute (wi th 0.1 minute accuracy) forcollection of vitamin D3

B. Analytical HPLC System-FIG. 3 for configuration.

1. Column:2. Eluant:

Hypersil APS II (4.6 x 250 mID, 311).1.0% l-chlorobutane + 0.5% amyl alcohol + 0.25%chloroform in i so -oc tane .

12-continued

3. Run Time:4.5.Approximately 35 minutes.1.5 mlImin.4O(1)" C.

Flow Rate:ColumnHeater:6. Detection:

7.264 nm @ 0.0025 AUFS, (Waters 486).Recommend the use of an integrator or data systemfor reprocessing.

8. Equilibrate th e columns and obtain a stable basel ine. Injectth e intermediate standard ISTD (no column switch) at least3 times unti l a consistent retention time (retention time 0.02minutes) is established on the cleanup HPLC (FIGS. 4 &5). Always veri fy the cleanup HPLC retention t ime within 'hhour before analysis of standards or samples. The run timeis approximately 20 minutes, however the t ime r e qu i re d toequilibrate th e columns with fresh eluant is approximately 2hours.After determining the retent ion time of the intermediate standardISID on th e cleanup column, set th e slice window (i,e. transfer ofvitamin D3 from th e cleanup column to the analyt ical column). Thisis done by setting the switching valve to switch the vitamin D3 fromthe cleanup column to the analytical column at 0.10 minutes beforethe vitamin D3 first e lu tes from the cleanup column unti l 0.10minutes after the vitamin D3 peak returns to baseline on th e cleanupcolunm. See FIGS. 4 and 5. Slice window times should notexceed 1.0 minute - using a minimum among of time (generally 0.8-1.0 min.) necessary to col lect a J J th e vitamin D3 while preventingth e transfer of any other interfering components.See FIGS. 6 &7 for the cleanup and analytical HPLCchromatograms of a 15T working standard.

Recorder:

15 9.

VI. HPLC ANALYSISA. Upon verifying equilibration of the HPLC system andestablishing the collection window, inject three (orfour) working standards (3T, ST, 15T, 3OT) and then thesample extracts. The three (or four) working standardsshould be injected once again at the end of the run.Only single injections of each sample are required.vn. CALCULATIONS (Use only peak heights for reportingpurposes)Note: Peak height is required for quantitation as smallamounts of baseline noise can cause large area differences.A. Calculation of Working Standard Concentrations1. Calculate the concentration of the vitamin D3 in work-ing standards 3T, 5T, 15T, 30T from the followingequation:

50

- (w) (p) (7) (pv) (W) (PV) (00112)IU/mI- (500)(500) (100) = .where: w = weight of vitamin D3 standard in mg.

P = 40,000 IU/mg for vitamin D3PV = final p ipet volume for working standards.

=3for3T= 5 for 5T.= 15 for 15T.= 30 for 3aI'.

Example: for a 5T standard prepared from a stock solutionthat contained 24.00 rn g vitamin D3, the concentrationis calculated as follows:55IU/mi (24.00) (40.000) (7) (5) 13440 IU/mi(500) (500) (100) =.

B. Calculation of the Standard Curve Using Linear60 Regression and the Quantitation of Vitamin D3 in Samples

1. The peak heights of each respective level of theworking standard are averaged. A linear regression lineis calculated by using the average peak heights (y-axis)and the concentration (x-axis) for the respective work-ing standard.Example: A linear regression line (vitamin D3 peakheights versus concentration) for 264 nm channel is

65


20/35

5,698,22213

presented below. Two injections (beginning and endof run) were made per each level of working stan-dan!.

Working Cone. No. Avg.PeakSid. IU/mI Inject Height

ST 1.3440 2 2.63%1 ST 4.0320 2 8.07893a T 8.0640 2 16.5839

Slope = 2.07775y-intereept = -0.20754Corr. Coef, = 0.99994

2. The samples should be quantitated by bracketing the 15standards around the samples.

C. Low pH: Beverage, and Vitamin D3 Emulsion, andPowder Product Calculation

1.Per Weight Basis-ill/Kg: Vitamin D3 (IUIkg)=(C) (V)(1000)# 20

(C) (V) (1000)#Vitamin D3 (IUJkg) (8) (X)

where: CeVitamin concentration (IU/mI) from standard 25curve.V=Volume (mI) of iso-octane to reconstitute extracts.1000=converts grams to kilograms.S=Sample size in grams.X=0.86 for 75 C. saponification factor for thermal 30isomerization of vitamin D3 to previtamin D3.#=Substitute 100 for 1000 to convert to IU/lOO g.

Example: A 12.533 g (S) low pH beverage sample wasreconstituted in 2ml (V) of iso-octane which generated 35a peak height of 6.2330. The corresponding vitamin D3concentration (C) obtained from the previously calcu-lated standard curve was 3.0998 ill/mI. The final con-centration would be calculated in the following man-ner:Vitamin D3 (IUJkg) = (3.0998) (2) (1000) =575 IUJkg(12.533) (0.86)

CALCIUM ASSAYThe Simultaneous Determination of Calcium (Ca) in a LowpH Beverage by ICP-AES Using a High Solids NebulizerA. THEORY1.Inductively coupled plasma atomic emission spectrom- 50etry (ICP-AES) is an atomic spectroscopic techniquethat has several advantages compared to atomic absorp-tion: excellent detection limits, a bread linear calibra-tion range of over four orders of magnitude for mostelements, minimal interferences, and the ability to 55determine several elements in the sample simulta-neously under one set of operating conditions. Theseadvantages translate into less sample preparation,calibration, and analysis time for the analyst.

2. The ICP-AES instrument consists of three components: 60sample introduction device, torchbox, and spectrom-eter. Most commonly, samples are introduced in theform of solutions which are nebulized (broken into tinydroplets), and passed into the torch with a stream ofargon. In the torchbox, 1-2 kW of radio-frequency 65power is coupled from a copper coil (inductor) into asmall region inside a quartz tulle (torch), through which

5

14argon flows. The power density in this region is highenough to heat the argon until it ionizes and, since theregion is at atmospheric pressure, there are sufficientcollisions with other argon atoms to instantly ignite aplasma with a temperature of about 10,000 K.

3. The micrometer-sized droplets from the nebulizer enterthe bottom of the torch and pass through the cooler(6000 K), darker, central region of the plasma called theaxial channel. Here water is evaporated, and theremaining dry particles of analyte are vaporized andatomized (molecules broken down into atoms) by theheat of the plasma injust a few milliseconds. Excitationand ionization of the outer electrons of the atomsoccurs; the intensity of the emission that results fromthe deexicitation of these atoms and ions is proportionalto the concentration of analyte in the original solution.Thus, calibration consists of measuring the intensity ofanalyte emission for standards of known concentration.

4. Light emitted by the ICP is collected by a lens in thespectrometer and focused onto a diffraction gratingwhich disperses the light into its component wave-lengths. The emitted radiation, wavelength resolved,from all the analyte elements is collected simulta-neously by several detectors placed in front of thegrating and converted into an electrical signal. A datasystem relates these signals to the concentrations of theelements in the standards and calculates the analyteconcentration in the samples.

5. The particular instrument used in this method featuresa movable entrance slit controlled by a high resolutionstepper motor called SAM! (Scanning Accessory forMultielement Instrumentation). Moving the entranceslit slightly changes the angle of incidence upon thegrating, and slightly changes the wavelengths incidentupon the exit sifts. This feature allows the user toperform background correction in the sample matrix bysubtracting the emission background just off the peakcenter.

6. This method employs a speedy dilution preparation ofsamples with a surfactant and dilute acid. A special kindof nebulizer called a maximum dissolved solids nebu-lizer (MDSN), or high-solids nebulizer, is required toprovide long term operation without clogging. Becausethe viscosity of standards and samples is quire different,an internal standard must be used to compensate for thepoorer nebulization efficiency of the high solidssamples. Cobalt is added to each standard so that theyare exactly 20.0 mgIL Co. Calibration consists ofmeasuring the analyte/Co ratio in the standards as afunction of analyte concentration. An exact quantity ofcobalt is added to each sample so that if they werediluted to 50.0 mL, their cobalt concentration wouldalso be 20.0 mgIL. Note, however, that the analytelinternal standard ratio in the samples will not changewith the total volume, and so volumetric ware is notnecessary for the sample preparation. When the soft-ware asks for the "sample volume" to calculate adilution factor, the analyst should enter 50 mL, thevolume that would make the concentration of cobalt inthe samples equal to that in the standards.MATERIALS

1. Instrumenta. Inductively Coupled Argon Plasma EmissionSpectrometer, ARL Model 3560 or Accurisb. Ryton V-groove nebulizer: ARL#173259-oo00 orPrecision Glass #510-50 only

c. Spray chamber: ARL#173142-oo03 or PrecisionGlass #110-34 or equivalent

10

40

45


21/35

d. ICP torch: ARL#139009-0003 or Precision Glass#l00-05 or equivalent

2. General Laboratory Equipment/Facilitiesa. Analytical balanceb. Fume hoodc. Disposable, flat-bottomed, 50 mL plastic centrifugetubes with caps (Baxter C3902-14 or equivalent)d. Plastic coated rack suitable for holding many cen-trifuge tubese. 125 mL, 250 mL, and 1L plastic bottles for storing 10standards: polymethylpentene (PMP) or equivalent --------------------

f. Disposable plastic transfer pipets-3.5 mL capacityg. Eppendorf pipet or equivalent, 1000 ~ capacity withtipsh. 50 mL repipetter or equivalent

i. Plastic dispenser bottle (PMP or equivalent) fittedwith a Teflon-constructed dispenser top with adjust-able volume between 1-10 mL; dispenser may befitted to concentrated HCI bottle directly

j. Magnetic stir plate and Teflon coated magnetic stir 20bars

k. 1L and 250 ml volumetric flasks: glass or plastic(PMP or equivalent)1. Class A volumetric flasks: 2,4,5,10,15,20,25,40,50mLm. Options: 1 mL digital pipet with tips, Rainin EDP-Plus or 1 mL, glass volumetric pipet or equivalent

3. Chemicals/StandardsUnless otherwise noted, the following chemicals should 30be stored at room temperature. Their expiration date isone year after the date they are first opened. Uponexpiration the chemicals must be either discarded orre-evaluated.a. High purity stock standard solutions (NIST or NIST- 35traceable) 10,000 mgIL Ca, 10,000 mg/L Co, 1000mg/L Co. These stock standard solutions expire onthe date given by the manufacturer.

b. Hydrochloric acid, J. T. Baker BIA-grade or equiva-lent 40c. Triton X-100, Kodak scintillation-grade or equiva-lentd. Argon gas, minimum 99.996% purity

e. High purity water, Millipore-treated or equivalentC. INSTRUMENfAL OPERATING CONDITlONS1.The wavelengths that have been used are listed in thetable below. The instrument should be installed withidentical channels ifpossible because the sensitivity ofthe line and the possibility of interferences can changeif a different line is employed for analysis. 50

5,698,22215

ELEMENT WAVELENGTII (run) TYPE ORDERCa ion 2317.93

2. Typical ranges of operating conditions for the ARL3560 are listed below.

a. Incident power: 1200-1400watts


22/35

5,698,22217

800 mL high purity water to a lL volumetric flask and20 mL (repipetter) of hydrochloric acid to the flask.Add 50 mL of Triton X-lOO solution from the dispenserto the flask and then fill the flask to volume with highpurity water, slowly to avoid forming suds. Agitate well 5and transfer to a clean, dry 1 liter storage bottle.Dispense as needed into 125 mL storage bottles to useat the instrument. Expiration: 6 months. Note that theISRB does not contain cobalt, but the standard blankdoes. The ISRB is analyzed before any standards or 10samples; the purpose is to subtract the intensity ofanalytes found in the reagents (Triton X-lOO solution,HCI, and water) from the analyte intensities found inthe standards and samples.

E. PROCEDURE1. Standard Handlinga. All bottles used for storage of standard solutionsmust first be soaked in 10% (v/v) HCl for a minimumof three hours, followed by multiple rinses with highpurity water. Air dry or rinse several times with the 20standard. When reusing the bottles for a new batch ofthe same standard, no acid soak is necessary-simply rinse several times with high purity water andthen several times with small portions of the freshstandard. 25b. As the working standards in the 125 mL bottles areused up, simply refill the bottles from the 1 Lstandards prepared in D. 3.

c. Because there are many samples, the most efficientway to add the cobalt internal standard to the 30samples is with a 1 mL digital pipet.

2. Sample Preparationa. Refill the reagent containers before preparingsamples so that the same batch of reagents can beused for all samples and the blank. 35

b. Remove the caps and arrange the empty 50 mL tubes,with labels, in the rack beginning with the sampleblank and two tubes for each sample.c. Transfer sample to a plastic storage container. Placethese containers directly on a magnetic stirring plate 40and add a Teflon coated stirring bar. Set the stirrer atan intermediate speed. After a minimum of oneminute of agitation begin to withdraw the sample forweighing with a disposable plastic transfer pipet.

d. Carefully weigh and record to the nearest 0.0001 g, 455 g of sample into the plastic tubes. The sample blanktube is left empty at this point. Add the followingreagents to each tube, including the blank, in thisexact order:(1) Add 2.5 mL of Triton X-lOO solution using the 50dispenser bottle.(2) Add approximately 45 mL of high purity water(3) Add 1.00 mL of the 1000 mg/L cobalt internalstandard with either a calibrated digital pipet(preferred) or a Class A pipet. 55(4) Add 1 mL of concentrated hydrochloric acid withan Eppendorf pipet or from a Teflon dispenserbottle.(5) Add high purity water until the total volume ineach tube is approximately 50 mL. Put the caps on 60and shake the tubes thoroughly.

3. Instrumental Analysisa. The following instructions refer to some generalcharacteristics of the PLASMA VISION software,which is currently used on all instruments running 65this method, but no attempt has made to descnbespecific key sequences needed to perform these

15

18procedures, since that information is provided intraining. Equivalent operations must be performedwith other versions of the software.

b. Turn on the plasma and allow a thirty minutewarm-up time before calibration. Turn on the com-puter and printer and start the software. Begin pump-ing 2% HCI rinse solution through the nebulizer.

c. Perform an instrument configuration before the firstcalibration is made for each 8-hour shift. This willcheck computer-instrument communications andcheck the SAM! motor. Watch the motor to be surethat it turns properly.

d. Check the optical alignment using the 150 mgILcalcium standard. This procedure will insure that theSAM! motor is operating properly and that thecalibration will always be performed near the exactcenter of each analyte peak. Perform this procedurebefore the first calibration is made, once during each8-hour shift. Choose appropriate setup options andthen run the profile. The measured peak centers forthe element to be measured must be within 6 SAM!units of the current SAM! profile position. If thisresult is not obtained, consult the supervisor: eithera new default SAMI profile position needs todefined, or the instrument requires service.

e. Select the appropriate task and the appropriate cali-bration sequence file name and begin calibration.Aspirate the standard solutions into the plasma start-ing with the ISRB solution prepared in D.5. Thesoftware prompts for each standard by name. Bealert to any error messages. Ifan error occurs, writedown the message and consult the supervisor. Afterthe last standard has been run, save the data and havethe software calculate a linear regression for eachelement. Print the calibration data, which summa-rizes the element intensities in each standard, thecorrelation coefficient, and the calculated concentra-tions of the elements in each standard.

f. Enter the section of the software to analyze thesamples. Set the print options to print whateverdocumentation is required. Select a name for the filethat will store sample results; if no file name ischosen, the program will store the data under the taskfile name by default.

g. Shake the samples immediately before introductioninto the ICP.

h. HIT THE ENTER OR REfURN KEY AFTERENTERING THE WEIGHT, VOLUME ANDNAME OF THE SAMPLE. Note that the PLASMA-VISION software prompts for the sample weight andvolume before the sample is introduced, and thesample name after it has been analyzed. The dilutionvolume for samples will always be entered as 50 mL,regardless of the actual volume in the sample tube.

i.Make sure that the sample introduction tube is placedin the 2% HCI rinse solution for at least 2-3 secondsbetween samples. Analyze the standards and thesamples in the following order:(1) Analyze the intermediate check standard solu-tions.

(2) Analyze the ''reagent blank" (or sample blank,contains cobalt). The intensities of the analytes inthis blank will be subtracted automatically fromthe intensities found in the samples.

(3) Analyze each sample in duplicate.j. Results can be reported in any convenient concen-tration units, depending upon how the tasks are


23/35

19programmed. Note: in cases in which the analyst hasentered the calibration standards into the task as"mgIL" and has entered the dilution volume as "50mL" and the sample weight in grams, the sampleresults will be in units of ug/g. The actual printout 5will show whatever units are programmed into thetask for each element The unit "ug/g" is preferred to"ppm" for reporting sample results because the latterterm is ambiguous. In laboratories where sampleresults for this method are commonly reported as 10"ppm", it must be understood that this really means"micrograms of analyte per gram of sample."

VITAMIN C (L-ASCORBIC ACID)DEfERMINATIONA. SAMPLE SIZE AND PRODUcr APPLICABILITYSamples should be as uniform and representative of theproduct as possible. Sampling should be performedimmediately after a gentle mixing or stirring to prevent

I 20inaccurate sampling due to stratification. All samp eweights must be recorded to at least three significantfigures.

Sample sizes for low pH beverages are calculated fromthe following equation.

Sampl e s iz e =35Q IEwhere:Sample Size is the theoretical sample size, in grams; E isthe expected ascorbic acid concentration in milligrams 30per liter or kilogram, respectively, as is, and; 350 is thedesired amount, in micrograms (meg), of ascorbic acidin the sample preparation. The net conversion factor formicrograms to milligrams and kilograms to grams isunity.B. THEORY

In this method the amount of L-ascorbic acid present inthe sample is determined by coulometric titration. Acoulometric method of analysis measures the quantityof electricity required to carry out a chemical reaction. 40If the reaction is 100% efficient, the passage of oneFaraday of electricity will cause the reaction of oneequivalent weight.

In this case, iodine is coulometrically generated fromiodide. The iodine then oxidizes the L-ascorbic acid to 45dehydroascorbic acid. When enough iodine has beenproduced to oxidize all the L-ascorbic acid in thesample, an excess of iodine will occur. This excess ofiodine signals the equivalence point, and is detected bytwo constant potential electrodes. The quantity of elec- 50tricity used is given by the product of current times thetime to reach the end point of the coulometric titration.Thus the amount of iodine used is equal to the numberof equivalents of L-ascorbic acid, and the amount ofL-ascorbic acid can be calculated. 55

Trichloroacetic acid is added to the sample to precipitatethe protein and to maintain the acidic condition neces-sary for a quantitative reaction.

C. APPARATUSAnalytical BalanceBeakers, 100 mI, graduatedBrinkmann E585 PolarizerCable for Double Platinum Wire Electrode, Brinkmanncat. no. 20-97-738-8, or equivalent

Cables for Platinum Foil Electrodes, Brinkmann cat. no.20-97-770-1 and 20-00-853-9, or equivalent

5,698,22220

15

Chart PaperDesiccatorDisposable PipetsDisposable tips for pipettorDouble Platinum Wire Electrode, Brinkmann cat. no.20-92-350-4, or equivalentElectrode Holder

Eppendorf pipet or equivalent, 200 melIsolation Tube, outer diameter 20 mm, 125 mm long, PoreSize C,Ace Glass Company cat. no. 7209-16; OR outerdiameter 12 mm, 125 mm long, Pore Size E, Ace cat.no. 7209-10. Size of isolation tube depends on size ofelectrodes used.

Keithly Model 225 Constant Current Source OR KeithlyModel 220 Constant Current Source

Magnetic Stir PlatePipettor; 5 rnl-Oxford, Wheaton, Eppendorf, orFinnipepette

Pipetrot or dispenser; 10 rnl and 30 rnl-Oxford,Wheateon, Lab Industries or equivalentPlatinum Foil Electrodes, (2), Brinkmann cat. no. 20-92-110-2, or equivalent

Sample vials, 5 mI with screw capsShields: yellow or clear shields with a cutoff of 385nanometers

Strip Chart Recorder; Kipp & Zonen or equivalentTeflon-coated Stir BarsUltrasonic Bath

25

Vacuum Flask, 2,000 mLVolumetric flasks, 100, 500 rnl, 1000 rnl with stoppersD.REAGENTS

I. CHEMICALS35 L(+) ascorbic acid (USP Reference Standard, OfficialLot); store in a desiccatorMetaphosphoric acid; ACS or equivalentPotassium iodide; ACS or equivalentSodium sulfate, anhydrous granular; ACS or equivalentTrichloroacetic acid; ACS or equivalent

II. SOLlITIONSNOTE: All solutions, samples and standards must beprepared and stored under UV shielded or yellowshielded lighting unless otherwise stated (seeAPPARATUS).

NOTE: Degassed water should be used for the O.lMpotassium iodide solution to prevent air oxidation of I-to 12 Degas water by placing deionized water into avacuum flask, and placing it under vacuum for 15minutes with sonication.

1. Potassium Iodide (0. 1M) Weigh 8.3 (O.5) g of potas-sium iodide into a 500 rnl volumetric flask. Dissolveand dilute to volume with degassed, deionized water.Store in a tightly stoppered brown bottle at roomtemperature. Do not store for more than one (1) weekDiscard if solution acquires a yellow tinge.

2. L-Ascorbic Acid Standard (2000 mgIL) Store standardbottle in a desiccator to prevent moisture absorption.Weigh accurately 0.200 (O.OOOS)g and transfer quan-titatively to a 100 rnl volumetric flask. Dissolve anddilute to volume with 3% metaphosphoric acid. Thestandard can be made fresh just before use each day, orcan be stored in small vials in the freezer. Standardstored in the freezer is good for two months.

3. Trichloroacetic Acid (1M) Weigh 163 (O.5) g oftrichloroacetic acid into a I liter volumetric flask Add

60

65


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5,698,22221

500 ml degassed deionized water. Swirl until dissolved,then dilute to volume. This reagent may be stored forone month at room temperature.

4. Sodium Sulfate (1M) Weight 142 (O.5) g of sodiumsulfate into a 1 liter volumetric flask. Add approxi- 5mately 750 ml of deionized water and mix until dis-solved. Dilute to volume with deionized water. Thisreagent may be stored for six months at room tempera-ture.

5. Metaphosphoric Acid (3%) Accurately weigh 15.0 10(O.lO) g of metaphosphoric acid and quantitativelytransfer to a 500 m1 volumetric flask. Add approxi-mately 250 ml deionized water and swirl until Meta-phosphoric acid is dissolved. Dilute to volume withwater. This solution may be stored for one week under 15refrigeration (2_8 C.).

E. PROCEDUREInstrument Settings-FIGS. 8, 9, 10 and 11.

a. Recorder Chart speed/input 1 mmlseeond or 5 emfmin,depending on chartreeorderll volt

b. Polarizer E585 Constant potential 150 mVsensitivity 10 mieroampse. Keithly 1.56 rn a 25Constant CurrentSource

1.Fill the isolation tube containing the cathode electrodewith 1M sodium sulfate. The sodium sulfate continu- 30ously moves from the isolation tube through the glassfrit into the sample solution and must be frequentlyreplenished.

2. An instrument check should be done at the start of eachday. Using an Eppendorf pipet (or equivalent), pipet 35200 microliters of the 2000 mgIL L-ascorbic acidstandard into a 100 ml beaker. Follow steps 5 through12 of the procedure. Follow steps 1 through 4 of thecalculations. Compare the experimentally determinedconcentration to the theoretical concentration. The 40results should be within 4% of the expected value. Ifnot, run another instrument check using fresh reagentsand standard. If the experimental result still differs bymore than 4% from the expected standard value, con-sult the method supervisor. 45

3. Agitate samples well before and during sampling.Liquid samples must be freshly opened. Analyses mustbe completed within 20 minutes after the container isopened. Samples are weighed directly into the beakerunless otherwise stated. 50

4. While swirling the sample, add 5.0 (O.l) ml of 1Mtrichloroacetic acid (TCA) to the sample. Swid for 30seconds to completely precipitate the protein.

5. Add 30.0 (O.5) ml of O.lM potassium iodide to the 55sample.6. Add degassed deionized water to approximately the 60ml mark.7. Add a stir bar. Lower the electrodes into the samplesolution. Make sure that all the electrodes are 60immersed. The sodium sulfate level in the isolationtube must be at least 2 em above the sample level.Adjust the magnetic stirrer speed so that stirring isvigorous but no air is entrained.

8. Switch the chart recorder on. Switch the polarizer on. 65Adjust the base line of the chart recorder to 10% of fullscale.

20

229. Switch the constant current source on.10. Titrate until excess iodine is produced, indicated by arising current curve. Stop the titration when the risingcurrent curve has reached at least 70% of full scale onthe chart recorder paper.

11. Switch the recorder chart speed to off and the constantcurrent source to standby.12. Remove the electrodes from the sample and rinse wellwith deionized water.F. CALCULATIONS

1.Extrapolate the linear portion of the rising current curveto the base line to locate the end point. The portion ofthe curve between 70% and 30% of full scale will belinear. (FIG. 12).

2. Count the number of centimeters from start of titrationto the end point to the nearest 0.1 centimeter.

3. Convert this distance to seconds of titration time.4. Calculate the amount of L-ascorbic acid present in thesample by the following formula:

C mXiXtnxsxF ixtxRs

Where:C=concentration of L-ascorbic acid in mgIl or mglkg(mcglml=mgIl and mcglg=mgIKg)

m= 176 (gram molecular weight of L-ascorbic acid)n=2 (change in valence)i=current in milliampst=time in secondsF=96,487 coulombs/equivalentR=proportionality constant for m,n and

F = 0.912 ....!!!:L-rnA-sec

s=sample size in gEXAMPLE: Assume a 3.0 g sample of a low pHbeverage was analyzed and the measured length oftitration on the strip chart was 19.0 em. Chart speedwas 1 mrnIsec. The current during the titration was1.56 rnA

c Rxtxisi= 1.56rnAs= 3.0 gR =0.912 meg/rnA-sec

t=~ x~ x 10mm =190see1 Imm lernc (1.56 rnA) x (0.912meg/rnA-sec)X (190 sec)3.0g

90mcglg90mglKg

SELECTION OF INGREDIENTS USED INPRACTICING THE INVENITONThe present invention provides high levels of calcium and

vitamin D in a carbonated beverage, a noncarbonatedbeverage, a liquid beverage concentrate, a powdered bever-age concentrate, a powdered beverage additive, beveragescontaining a powdered beverage concentrate or additive, ora calcium supplement. As used herein and in the claims theterms "liquid nutritional product" and "beverage" are under-


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5,698,22223

stood to be synonymous. As used herein and in the claims a"low pH beverage" is understood to refer to a beveragehaving a pH of less than 4.6. Trial batches of low calorielemon lime, orange, peach, and wild cherry flavored proto-type carbonated beverages have been manufactured in 5accordance with the present invention. The prototype bev-erages were manufactured by preparing a beverageconcentrate, then blending the beverage concentrate withtreated water. The blends where then carbonated and filledinto standard 12 ounce soda aluminum cans. (Soda alumi- 10num cans are coated in accordance with accepted industrystandards to substantially reduce migration of aluminum intothe contents of the can.)Calcium Source. As used herein and in the claims the term"calcium" used alone refers to elemental calcium, the term 15

"calcium salt" refers to a chemical composition containingelemental calcium, and "calcium source" refers to calciumand/or a calcium salt. The calcium salt used in preferredembodiments of the present invention is Calcium Glycero-phosphate (CaGP) which is generally recognized as safe 20(GRAS) by the United States Food and Drug Administration(FDA) (21 CPR 170.3). Another reason for selecting CaGPis that, as already disclosed above in the background section,it is one of the ten calcium compounds recognized by FDAas safe and lawful for use in a dietary supplement or as a 25nutrient supplement for osteoporosis. However; any othersuitable calcium source, such as calcium citrate malate thatwould be soluble at a pH of about 3.5-4.5 could beemployed in the practice of the present invention.Calcium glycerophosphate (CaGP) can be described as a 30

white, odorless, almost tasteless powder. Its solubility inwater increases in the presence of citric and lactic acids, asstated in the Merck Index. The CaGP used in the trial batchesw as FCC ill grade a nd w as p ro du ced by D r . Pau l LohmanGmbH, Emmerthal, Germany and is distributed by Gallard 35Schlesinger Industries, Inc., Carle Place, N.Y., 11514, USA.Another reason for selecting CaGPis its excellent calcium

bioavailability. Churella et al., "RELATIVE CALCillM(CA) BIOAVAll.,ABll.ITY OF CA SALTS USED ININFANT FORMULAS", THE FASEB JOURNAL,4(3) 40:A788 (1990) reports a study which determined the calciumbioavailability of four calcium salts. Rats were fed variousdiets containing different calcium salts for three weeks. Atthe end of the study, the right femur was removed and testedfor calcium. As compared to a control, the relative calcium 45bioavailability was as follows: tricalcium phosphate 110%,calcium citrate 110% and CaGP 106%. Furthermore, studiesreported by Hanning et al, "Efficacy of calcium glycero-phosphate vs conventional mineral salts for total parenteralnutrition in low-birth-weight infants: a randomized clinical 50trial"?", AMERICAN JOURNAL OF CLINICALNUfRITlON, 54: 903-908 (1991), and Draper et. al., "Cal-cium Glycerophosphate as a Source of Calcium and Phos-phorous in Total Parenteral Nutrition Solutions", JOURNALOF PAREN1ERAL AND ENTERAL NUTRITION, 15(2): 55176-180 (1991) showed in low birth weight infants andpiglets, respectively, that CaGP is as effective as calciumgluconate as a source of calcium in total parenteral nutrition(TPN) solutions and could be used to prevent under miner-alized bones in low birth weight infants.Yet another reason for selecting CaGP was its high

solubility which facilitates a larger calcium intake per serv-ing. A number of calcium salts were evaluated for theirfunctionality in the liquid nutritional product of the presentinvention: dicalcium phosphate, monocalcium phosphate, 65calcium chloride, tricalcium phosphate, calcium citrate, cal-cium carbonate, CaGP, and D-gluconic acid (hemicalcium

24salt). Aqueous solutions containing 500 mg of calcium per237 mL (8 oz.) serving (2110 ppm) were prepared and thepH was adjusted to pH 3.5 and pH 5.0. Results indicated thatsolubility of calcium salts varied and only calciumcarbonate, calcium chloride, CaGP, and D-Gluconic acid,remained soluble at pH 3.5 for at least one month. In thisevaluation solubility was determined by a visual examina-tion. At pH 5.0 all samples formed crystals over time. Theresults of this solubility study are presented in Table 3.

TABLE 3SOLUBILITY OF CALCIUM SOURCES(Solutions at 500 mp; calcium per 237 mLl

At Time of Manufacture 1 MONTIISalt pH 3.5 pH 5.0 pH 3.5 pH 5.0Dicalcium insoluble insoluble insoluble insolublePhosphateMonocalcium insoluble insoluble insoluble insolublePhosphateCalciwn soluble soluble soluble insolubleChlorideTricalciwn insoluble insoluble insoluble insolublePhosphateCalciwn insoluble insoluble insoluble insolubleCitrateCalciwn soluble partially soluble insolubleCarbonate SolubleCaGP soluble soluble soluble insolubleD-Gluconic- soluble soluble soluble partiallyAcid* Soluble*Hemicalciwn saltExperiments were repeated with calcium carbonate,CaGP, a nd c alc ium c hlo ride in a c om ple te liqu id n ut rit io na l

product matrix, i.e., in conjunction with aspartame, a flavorsystem and vitamin C. The pH range evaluated was 3.5-4.5.At the lower end of the pH range all calcium sources weresoluble at time of manufacture. After one month it wasobserved that as the pH increased, calcium carbonate andCaGP formed crystals, worse in the case of calcium carbon-ate. In addition, it appeared that the CaGP had a synergisticeffect with aspartame regarding sweetness. Calcium chloridewas completely soluble throughout the pH range but itsbitter flavor made it unacceptable for the liquid nutritionalproduct of the present invention application. Calcium lactatewas evaluated in subsequent experiments. Although itssolubility was excellent itprovided astringent and mineralsalt-type notes to the taste of the beverage that made itundesirable.Still another reason for selecting CaGP is the fact that abeverage matrix containing this calcium salt requires theaddition of less acid to achieve a pH below 4.0. Acidity isdesired in the liquid nutritional product of the presentinvention for several reasons such as: to maintain thecalcium salt solubility, to complement flavor, to controlmicrobial growth, and to enhance the role of preservatives,specifically potassium benzoate or sodium benzoate. On theother hand, too much acidity can result in increased tartnessand sourness that make the product undesirable from a

60 sensory point of view. When calcium salts are added to theliquid nutritional product of the present invention, the solu-tion resists changes in pH and more acid is needed to bringdown the pH than in commercially available sodas with nocalcium fortification. Aqueous solutions of various calciumsalts were prepared to deliver 500 mg of elemental calciumper 12 oz. serving (1408 ppm) and the pH adjusted to pH 3.5with citric acid. Titratable acidity was determined by mea-


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25suring the amount of O.lN NaOH needed to raise the pH to8.3 in a 40 g sample containing 1,409 mglKg of a calciumsource. The results presented in TABLE 4 indicate that, withthe exception of calcium chloride, CaGP was the calciumsalt that had the lowest titratable acidity. Titratable acidity isan indication of the total acidity of a beverage.

TABLE 4TIfRABABlE ACIDITY OF CALCIUM SOURCES

Calcium SourceTitratable aciditymL of O.IN NaOH

Calcium ChlorideCaGPCalcium LactateTrica1cium PhosphateCalcium Citrate MalateCalcium CitrateCalcium HydroxideCalcium Carbonate

0.743.547.148.653.257.560.661.4

Calcium glycerophosphate (CaGP) is created by the reac-tion of glycerophosphate, a weak acid with pKf=6.1, withthe strong base calcium hydroxide. GaGP binds calciumwith an approximate formation constant of 1.7. CaGP, whendissolved in water, dissociates readily to provide "free" 25calcium ions and protonated glycerophosphate species.Acid-base buffering by monoprotonated glycerophosphate iseffective only within the pH range from 4.1 to 8.1 (refer tothe Henderson-Hasselback equation), and thus, GP exhibitsinsignificant buffering capacity at pH=3.6. On the other 30hand, anions, such as malate, tartrate, propionate orsuccinate, do provide buffer capacity at pH=3.6, and accord-ingly require more base or acid than GP for final adjustmentof pH.Yet another reason for selecting CaGP is the low alumi- 35num content in commercially available CaGP. It has beentheorized that chronic use of calcium supplements whichhave significant aluminum contents may constitute unnec-essary metal exposure. Whiting, "Safety of Some CalciumSupplements Questioned", NUTRITION REVIEWS, 52(3):95-97 (1994). The aluminum content of some calciumsources is presented in TABLE 5.

TABLESALUMINUM CONTENT OF CALCIUM SOURCES

Calcium SourceAlmninum Content

in parts per million (ppm)CaGPCalcium HydroxideCaC03 (from fossil shell)CaC03 (from Dolomite)

4.55300-400

4 , 4 O < f171-3152

'Values determined by analysis of commercia lly avai lable compounds.2Values from Whiting article.It has been suggested that calcium citrate may playa rolein enhancing aluminum absorption from food, potentiallyresulting in toxic serum and urinary aluminum levels.Sakhaee et al., have successfully demonstrated however, thatthe provision of calcium citrate alone without aluminum-containing drugs does not pose a risk of aluminum toxicityin subjects with normally functioning kidneys. Sakhee et al. ,

"Calcium citrate without aluminum antacids does not causealuminum retention in patients with functioning kidneys,"BONE AND MINERAL, 20: 87-97 (1993).Vitamin D. As used herein and in the claims the terms"vitamin D" and "various forms of vitamin D" are under-stood to refer to vitamin D, cholecalciferol (D3), ergocal-

5,698,22226

ciferol (D2) and its biologically active metabolites andprecursors such as, 10:,25-(OHh vitamin D; 25 OH vitaminD, its biological precursor; and 1 a, hydroxyvitamin D, andanalogues of the dihydroxy compound. These materials5 promote intestinal absorption of calcium, contribute toplasma calcium regulation by acting on the remodelingprocesses of accretion and resorption and stimulate reab-sorption of calcium by the kidney. While the form of vitaminD3 used in the following examples, prototypes and experi-

10 ments is cholecalciferol, it is understood that any of thevarious forms of vitamin D may be used in practicing thepresent invention, but vitamin D3 is preferred in embodi-ments which are liqnids.Dietary calcium and vitamin D are the natural mediators

15 against bone loss. Vitamin D acts directly on bone cells(osteoblasts, osteoclasts) to alter bone mass. It also promotesgut uptake of calcium. Human skin activates pre-vitamin Dmolecules when exposed to ultra violet irradiation. In thesummer, 15 minutes exposure to sunlight is sufficient tomaintain adequate vitamin D levels. On the other hand,

20 during winter, all day exposure to sunlight will producenegligible conversion of vitamin D. The thinner skin asso-ciated with aging is a less effective converter than theyouthful skin.The addition of vitamin Dto the liquid nutritional productof the present invention was difficult because this is an oilsoluble vitamin whereas both the beverage concentrate andthe beverage of the present invention are aqueous solutions.A number of possible methods to overcome the immiscibil-ity of these two phases were evaluated. The results of theseefforts are related below, and batch numbers are sequentialthroughout the following studies.There were two major obstacles to overcome regardingthe incorporation of vitamin D3 in the present invention: (t)the initial processing loss of vitamin D3; and (2) the stabilityof vitamin D3 over the shelf life of the product. To comparethe initial processing loss and stability of vitamin D3 Of eachvariable with successive batches, two criteria were routinelymeasured: (1) % recovery of vitamin D3 at O-time; and (2)half life of vitamin D3 (t1l2).40 The % recovery of vitamin D3 of each batch was calcu-lated by dividing the O-time vitamin D3 result by thetheoretical fortification of each batch times 100%. (SeeTable 6). As used herein ' 'theoretical fortification" refers toamount of vitamin D3 added to the product. As used herein

45 "O-time" refers to the time of initial vitamin D3 analysis ofthe product. In Table 6, "% Recovery" is the percentage oftheoretical fortification of vitamin D3 remaining in theproduct after initial processing loss. Only batch 31 did nothave the O-time vitamin D3 determined. Therefore, a pro-

50 jected result for this batch was extrapolated from the nega-tive exponential regression curve generated from the stabil-ity data.

TABLE 655 O-TIME VlTAMIN D3 RESULTS VERSUS

THEORKITCALFORITHCKaONTHEORET- %

BATCH O-TIME ICAL RECOVERY60 1 440 950 46.3%

2 405 950 42.6%3 450 950 47.4%

Mean for batches 45.4%1-365 4 249 635 39.1%

5 283 635 44.6%


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27TABLE 6-continued

O-TIME VITAMIN D3 RESULTS VERSUSTIffiORETICAL FORTIFICATION

TIffiOREf- %BlITCH O-TIME ICAL RECOVERY

6 294 633 46.4%Mean for batches 43.4%~

7 371 483 76.8%8 328 634 51.7%9 308 618 49.8%

Mean for batches 59.4%7-9

10 548 841 65.2%11 696 844 82.5%12 680 843 80.7%13 691 844 81.9%14 546 842 64.9%15 649 845 76.8%16 679 844 80.5%17 681 844 80.7%

Mean for batches 76.7%10-17

18 752 916 82.1%19 678 915 74.1%20 802 916 87.6%

(control batch)21 784 917 85.5%22 491 917 53.5%23 796 916 86.9%24 798 916 87.1%

Mean for batches 78.2%18-19 & 21-24

25 473 826 57.3%26 526 825 63.8%27 539 825 65.3%28 633 825 76.7%29 517 793 65.2%

(control batch)30 576 823 70.0%

Mean for batches 66.6%25-28 & 30

31 786 840 93.6%No Data Available -ExtrapolatedFrom the Exponential Regression Curve

To better characterize the stability of vitamin D3over timein all the batches, the Henri-Michaelis-Menton exponentialequation was employed. The vitamin D3 results (IU/KG) foreach variable was plotted versus time (Day) and a regressioncurve was fitted through the data using the following equa- 50tion:

Where:[D]=Vitamin D3 concentration (WIKG) at time (t).[Do]=Vitamin D3 concentration (IDIKG) at O-time,e=Exponentialk=Rate constant (rate of loss of vitaruin D3 over time)t=Time (days)Stability was defined as the amount of time (days) that 60

would be required for the initial concentration of vitamin D3to be reduced one half. This was termed half-life (t1l2). Themore stable the vitamin D3 in a particular formulation, thelonger it would take for the initial concentration to bereduced by one half. Rearranging the previous equation and 65making the appropriate substitutions, the half-life of vitaminD3 in a particular variable could be expressed as:

5,698,22228

tll2=In 2/kt1l2=Time (days) required for vitamin D3 to be reduced byone half of the initial concentration.In=Natural log.k=First order rate constant (rate ofloss of vitamin D3 overtime).The various batches are described in the following text.For convenience, the batch numbers are sequential. Inaddition, the actual vitamin D3 data at each time point for

10 each respective variable are presented in Tables 8, 10, 12,13, 14, 16 and 17. The correlation coefficients, initial vita-min D3 concentration [Do]' first order rate constants (k), andvitamin D3half lives (tllz) are also presented in Tables 8,10,12, 13, 14, 16 and 17 and should be referred to during the

15 discussion.A detailed discussion of each variable will not be pre-sented since such a presentation would be quite lengthy.Rather an overview of various batches grouped with respectto the main variables that were studied will be discussed.a. Use of a Water Dispersable Form of Vitamin D3Early in the development of the present invention anevaluation was made of a water dispersable vitamin D3spray dried on a dica1cium phosphate and gum acacia carrier.

The water dispersible vitamin D3 used in this evaluation wasa DRY VITAMIN D3 Type 100-DS purchased from Roche25 Vitamins and Fine Chemicals, a division of Hoffman-LaRoche, Inc., Nutley, N.J., U.S.A., which contains vitaminD3 (cholecalciferol USP-FCC), dicalcium phosphate, gumacacia, coconut oil, BHT, lactose, silicon dioxide, sodiumbenzoate and sorbic acid. Itis a white powder and contains30 100,000 Wig of vitamin D3.Three batches were manufactured to evaluate the waterdispersible form of vitamin D3. Each batch consisted of anaqueous solution containing potassium benzoate, citric acid,sodium citrate, aspartame, calcium glycerophosphate and35 the water dispersible form of vitamin D3. The resultantproduct was not homogenized. The final pH of each batch ispresented in Table 7. This pH difference, however, did notseem to affect vitamin D3 recovery.

5

20

TABLE 7~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -BATCH pH123

3.504.194.97

45 ----------------------~------~-------The initial processing losses for batches 1-3 was severe(mean=45.4% Recovery-Table 6). The loss of vitamin D3

was primarily due to: (a) the fact that the vitamin D3 was nothomogenized into the product matrix; and (b) there was noemulsifier present that would assist in maintaining thevitamin D3 in solution. Therefore, the vitamin D3 was lost bythe coating of the manufacturing equipment with vitaminD3. The stability of Vitamin D3 in these three batches wasnot acceptable over the shelf life of the product. As shown55 in Table 8, one half of the initial vitamin D3 was lost inapproximately 12.6 days.

TABLE 8Vl11\MIN D3 QUIKG OF PRODUC'!) VERSUS DAYS

BATCH 2 3

~5315210

~328191

450347225


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b. Use of Polysorbate 80 as an EmulsifierA series of experiments were conducted using vitamin D3in Polysorbate 80 manufactured to selected specifications byVitamins Inc., Chicago, Ill., U.S.A. Polysorbate 80 is a watersoluble, non-ionic emulsifier used for various applications in

the food industry. It is a polyoxyethylene derivative of 20 ---------------------sorbitan monooleate which interacts with the oil and aque-ous phases in an emulsion to form a barrier at the interfacethat causes a reduction in Van der Waals forces and animprovement in emulsion stability. Itwas expected that theuse of Polysorbate 80 to incorporate the vitamin D3 would 25improve its recovery and stability by causing dispersion ofthe oil phase in the continuous aqueous phase.The effect of Polysorbate 80 was evaluated in three

experimental batches of a low pH beverage. Liquid beverageconcentrates were prepared as described above, i.e., adding 30to water sodium benzoate (instead of potassium benzoate asa preservative), citric acid, potassium citrate, aspartame,calcium glycerophosphate, and vitamin D3 in a premixcontaining Polysorbate 80 and propylene glycol. The result-ant liquid beverage concentrates were not homogenized and 35were diluted with five parts of water before carbonation. Thevitamin D3 fortification level for each batch was 635 IUIKGof product All batches contained vitamin C. The variablesin batches 4-6 are presented in Table 9.These variables wereadded in an attempt to improve vitamin C stability, since it 40has been found that cysteine, when added in a carefullycontrolled amount can overcome vitamin C deterioration inpackaged beverages (U.S. Pat. No. 3,958,017, May 18,1976).

5,698,22229

TABLE 8-continuedVffi\MIN D3 aulKG O F PR OD UC T) V ER SU S D AY S

BAlCH 2 3Co rr . C o e f. 0.955 0.968 0.965[Dol 462 418 466k 0.0636 0.0501 0.0529t1 /2 10.9 13.8 13.1

A ve ra ge H alf L ife (t1l2) o f V it am in D 3 fo r B at ch es 1-3 is 12.6Days'D ay s a ft er i nit ia l v it am in D 3 t es tin g. O - tim e t es tin g o cc ur re d 7 d ay s a ft er th e~ r o du c t w a s m a nu fa ct o re d.R e su lt s f or b at ch e s 1-3 w ere c orre cted v ia co ntro l v alu e o n day 0 and day7;

TABLE 9BATCH VARIABLE456

C ys te in e, 1 .5 % o f V i!. CN o C ys te in eCysteine + 250 PPM W PC

The overall mean % Recovery for batches 4-6 wascomparable to the previous batches containing the waterdispersible form of vitamin D3. The mean % Recovery was43.4% (Table 6). However, as shown in Table 10, thestability of these batches improved significantly. The halflife of vitamin D3 in these batches ranged from 257 days to1,160 days. Cysteine addition did not affect vitamin D3recovery, but batch 6 with whey protein concentrate (WPC)showed minimal loss of vitamin D3 during 60 days of shelflife.In addition, batch 6 also had slightly better initial vitaminD3 Recovery than those batches in this series withoutprotein. This suggested that a more rugged emulsion andsome sort of matrix was needed as shown in Table 10. Theuse of WPC is not indicated if the product of the invention

30is desired to be low in calories or free of calories, butotherwise may be used in the practice of the invention. In anattempt to make a low calorie or calorie free product, the useof mechanical means such as homogenization was investi-

5 gated.TABLE 10

V IT AM IN D 3 aulKG O F PR OD UC T) V E RS US D AY S10 BAlCH 4 5 6

Days '0 249 283 2947 226 272 28225 243 243 28160 261 239 279C a rr . C o e f. 0.450 0.783 0.512[Dol 236 275 288k 0.0015 0.0027 0.0006t1 l2 462 257 1160

15

A verage H alf L ife (t1/2) o f V itam in D 3 fo r B atches ~ is 626 Days.'D ay s a ft er i nit ia l v it am in D 3 t es tin g. O - tim e t es tin g o cc ur re d 2 d ay s a ft er th ep r o du c t w a s m a nu fa ct u re d.C. Use of HomogenizationIn the next series of studies, the vitamin DJPolysorbate

premix was combined with the aqueous phase and the blendwas emulsified by passing it through a two-stage Gaulin-L-100 homogenizer at a given pressure. The purpose of thishomogenization step is to break up, or evenly disperse, theoil phase into the aqueous phase so that the particle size ofthe emulsion is sufficiently small to retard coalescence of theoil phase and prevent separation. A two-stage homogeniza-tion is needed since the fine particles formed during the firststage can clump. The second stage, set at a lower pressure,is needed to break up the clumps, thereby making a morestable emulsion.Brominated vegetable oil (BVO) and small quantities of

gum arabic were added to the vitamin DiPolysorbate pre-mix prior to homogenization. This was done to increase thespecific gravity of the oil phase and avoid phase separation,or oiling-off, of the emulsion. BVO is used in the sodaindustry as a stabilizer for flavoring oils used in fruitflavored beverages. BVO is a Food Additive (21, CPR

45 18030) allowed in an amount not greater than 15 ppm of thefinished beverage.A series of experiments were conducted to evaluate the

effect of homogenization on vitamin D3 recovery and sta-bility. In these experiments Iiquid beverage concentrates50 were prepared as described above with the exception of the

vitamin D3 addition. All the water soluble components werefirst dissolved in water and a vitamin D3 emulsion, preparedseparately, was added at 1% of finished productconcentration, and mixed thoroughly. The vitamin D3 ernul-

55 sion was prepared by combining water, vitamin D3 and oneor more of the following ingredients: Brominated VegetableOil (BVO), Polysorbate 80, Gum Arabic (GA), and corn oil,followed by homogenization using a two stage homogenizer.

60 Two different sources of vitamin D3 were used: (a) an oilsoluble vitamin premix where the vitamin D3 is dissolved ina small amount of corn oil; and (b) a vitamin D3 premixwhere the vitamin D3 is dissolved in Polysorbate 80 andpropylene glycol (PO) (same as batches 4 through 6). One

65 part of the complete concentrate was then dissolved withfive parts of water before carbonation. The variables inbatches 7-24 are presented in Table 11.


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31TABLE 11

VariableBatch (ppm = ppm of product)7 BVO, Vitamin D3 in Com Oil, GA (0.14 ppm)8 BVO, Com oil, Vitamin D3 in Polysorbate 80, GA (0.14 ppm)9 BVO, Com Oil, Vitamin D3 in Polysorbate 8010 BVO, Vit D3 in Com Oil, Polysorbate 80 (0.07 ppm), GA

(0.14 ppm)11 BVO, Vit D3 in Com Oil, Polysorbate 80 (0.035 ppm), GA

(0.14 ppm)12 BVO, Vit D3 inCom Oil, Polysorbate 80 (0.035 ppm), PG

(0.15 ppm), GA (0.14 ppm)13 BVO, Vit D3 inCom Oil, Polysorbate 80 (0.07 ppm), GA

(0.14 ppm)14 BVO, Vit D3 inCom Oil, Polysorbate 80 (0.07 ppm), PG

(0.30 ppm)15 BVO, Vit D3 inCom Oil, Polysorbate 80 (0.035 ppm)16 BVO, Vit D3 inCom Oil, Polysorbate 80 (0.035 ppm), PG

(0.15 ppm)17 BVO, Vit D, in Com Oil, Polysorbate 80 (0.07 ppm)18 Same as 1119 Same as 2220 Same as 1321 Same as 1522 Same as 1623 Same as 1724 BVO; Vit D, inCom Oil , Polysorbate 80 (0.07 ppm), Fructose

(42,000 ppm)

The gum arabic used in all batches was Nutriloid GumArabic from Tic Gums, Inc. When extra Polysorbate 80 wasadded to the batches, the percent addition refers to percentof oil in the batch. Batches contain either 3% or 6% extraPolysorbate 80 added. The 20% and 40% refer to combina-tions of Polysorbate 80 and Propylene glycol where thePolysorbate 80 content is 3% and 6%. Fructose was addedto batch number 24 to see if it would extend the shelf-life of

5,698,22232

improved the initial recovery and stability of vitamin D3versus previous attempts.

TABLE 125VITAMIN D, (IUIKG OF PRODUCl) VERSUS DAYS

BATCH 7 8 910 -----------------------------------------

Days'

15o72670Corr. Coef,

371 328 308354 372 346217 235 224189 284 236

0.816 0.224 0.506349 324 309

0.0098 0.0030 0.004771 231 147

[Dol20 k

Average Half Life (t,d Of Vitamin D3 for batches 7-9 is 150 days.25 'Days after ini tial v itamin D, testing. O-time testing occurred 8 days after the

product was manufactured.The vitamin D3 results for batches 10-17 confirmed that

homogenization was necessary. The mean % Recovery for30 these batches dramatically improved to 76.7% versus all

previous batches (fable 6). The overall vitamin D3 stability(mean half-life=68.6 days) for batches 10-17, as presentedin Table 13, was not as good as batches 7-9 (150 days) butwas superior in comparison to batches 1-3 (12.6 days).

TABLE 13VITAMIN D3 (lUIKG OF PRODUCT) VERSUS DAYS

BATCH 10 11 12 13 14 15 16 17Days'0 548 696 680 691 546 649 679 68110 484 469 47832 451 456 437 403 302 445 467 44157 250 382 351 321 228 355 358 34291 171 269 271 225 175 297 303 274Corr. Coef. 0.953 0988 0.931 0.948 0.956 0.966 0.963 0919[Dol 591 672 598 602 497 615 645 594k 0.0136 0.0102 0.0090 0.0111 0.0124 0.0086 0.0090 0.0090t1l2 51.0 67.9 77.0 62.4 55.9 80.6 77.0 77.0Average Half Life (t1l2) of Vitamin D3 for batches 10-17 is 68.6 days.'Days after initial vitamin D3 testing. O-time testing occurred 4 days afte r th e product wasmanufactured.

the product which is limited by the degradation of aspar-tame. In general, the fructose and the various levels of 55Polysorbate 80 did not affect the vitamin D3 recovery as thehomogenization step did.The initial vitamin D3 Recovery (mean=59.4%) and the

mean half-life value (150 days) for batches 7-9, as presentedinvitamin D3 Recovery Table 6 and Table 12, indicated that 60with few exceptions, the homogenization step signillcantly

In order to confirm the initial vitamin D3 Recovery andstability of batches 10-17, duplicate batches were made (seebatches 18-19 and 21-24 in Tables 6 and 14). The initialvitamin D3 Recovery for batches 18-19 and 21-24 (mean=78.2%) corroborated previous recoveries for batches 10-17.Furthermore, the vitamin D3 stability of batches 18-19 and21-24 (mean half-life=76.7 days) was comparable to theirrespective duplicate batches (68.6 days).


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5,698,22233 34

TABLE 14VITAMIN Da (lliIKG OF PRODUCD VERSUS DAYS

BATCH 18 19 20 21 22 23 24Days'0 752 678 802 784 491 796 79818 586 491 584 571 357 585 57763 379 309 358 381 245 372 40684 366 303 342 365 214 341 370Corr. Coef. 0958 0.938 0954 0.942 0.969 0.967 0.951IDol 714 627 747 725 458 746 738k 0.0088 0.0097 0.0103 0.0090 0.0095 0.0100 0.0088t1/2 78.8 71.5 67.3 77.0 73.0 69.3 788Average HaIfLife (t1l2) of Vitamin D3 is 74.7 Days. (Batch 20 is control and is not included inth e average.'Days af ter initial vitamin D3 testing. O-time testing occurred 1 day after the product wasmanufactured.

Although the shelf life data for batches 10-17 and 18-24showed a loss of vitamin D3 as a function time, no signifi- 20cant amount of degradation product could be analyticallydetected. Therefore, the main mechanis

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