Note: Descriptions are shown in the official language in which they were submitted.
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CALCIUM SUPPLEMENTS AND CALCIUM CONTAINING BEVERAGES COMPRISING VITAMIN D
FIELD OF THE INVENTIQN
The p,t:senl invention relates to calcium supplements and, in particular, to a solid
sl~FF'Pmer,l fortified with calcium glycerophosphdle, vitamin D and vitamin C; to a
beverage concenl, ~te or additive (liquid or powder) containing calcium and vitamin D;
and to a beverage made by reconstituting such beverage concer,l,ales and additives to
make a liquid nul,iliol1al product forliried with both calcium and vitamin D, and p,~r~rably
having a low pH.
BACKGROUND OF THE INVENTION
Calcium is an esse"lial nutrient; it is a major co",ponent of mineralized tissues
and is required for normal growth and dcvelopn,ent of the skeleton and teeth. Over the
last decade calcium has enjoyed increased attention due to its potential role in the
prevention of osteoporusis. Osteoporosis affects more than 25 million people in the
United States and is the major underlying cause of bone fractures in postmenopausal
women and the elderly. "Optimal Calcium Intake", JOURNAL OF THEAMERICAN
MEDICAL ASSOCIATION, 272(24): 1942-1948 (1994).
As used herein "osteoporosis" refers to a reduction in the amount of bone mass.
Two important factors influencing the occurrence of osteopol usis are optimal peak bone
mass attained in the first two to three clec~des of life and the rate at which bone mass is
lost in later years. Adequate calcium intake is critical to achieving optimal peak bone
mass and modifies the rate of bone mass loss associated with aging. Wardlaw, "Putting
osteoporosis in perspe~;ti~/e", JOURNAL OF THE AMERICAN DIETETIC
ASSOCMTION, 93(9): 1000-1006 (1993).
Several cofactors modify calcium balance and influence bone mass. These
include dietary constituents, hormones, drugs, and the level of physical activity. Unique
host characteristics may also modify the effects of dietary calcium on bone health.
These include the individual's age and ethnic and genetic background, the presence of
gastrointestinal disorders such as malabsorption and the post~a~ ctomy sylldlullle~
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and the plesence of liver and renal rlice~se. Interactions among these diverse cur~-ilur~
may affect calcium balance in either a positive or negative manner and thus alter the
optimal levels of calcium intake. "Optimal Calcium Intake", JOURNAL OF THE
AMERICAN MEDICAL ASSOCIATION, 272(24): 1942-1948 (1994).
Calcium requirements vary throughout an individual's lifetime with greater needsoccurring during the period of rapid growth in childhood and adc'os~ence, pr~y"ancy
and lactation, and in later adult life. Table 1 pr~senl:~ the optimal calcium requirements
which were ~e~ ' shed at a National Institute of Health (NIH) conrelence on optimal
calcium intake held June 6-8,1994. "Optimal Calcium Intake", JOURNAL OF THE
AMERICAN MEDICAL ASSOCIATION, 272(24): 1942-1948, at 1943 (1994). The
pa~ anl:, at the NIH co"r~rt:"ce considert:d former Recommended Dietary
Allowances (RDA) (10th edition,1989) for calcium intake as lererence levels and used
them as guideli"es to determine optimal calcium intake in light of new data on calcium-
related disorders.
TABLE 1: OPTIMAL CALCIUM INTAKES
OPTIMAL DAILY INTAKE
GROUP (in mg of calcium)
Infants
Birth-6 months 400
6 months-1 year 600
Children
1-5 years 800
6-10 years 800-1,200
A~lolescenls/Young Adults
11-24 years 1,200-1,500
Men
25-65 years 1,000
Over 65 years 1,500
Women
25-50 years 1,000
Over 50 years (postmenopausal)
On esllogens 1,000
Not on estrogens1,500
Over 65 1,500
P, ~yl lal ll and nursing1,200-1,500
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National consumption data indicate most females over the age of eleven, as well
as elderly men, consume amounts of calcium below recommended levels. "NationwideFood Consumption Survey, Continuing Survey of Food Intakes of Individuals", USDANFCS, CFS 11 Report No. 86-93 (1988). Accoldil ,g to the Second National Health and
Nutrition Exdr, li. ,alion Survey, the median daily calcium intake for women in the United
States was 574 mg. DIETARY INTAKE SOURCE DATA: UNITED STATES, 1976-80,
Data From the National Health Survey, Series ll, No. 231, DHHS Pl~lic;31ion No. (PHS),
pages 83-1681 (1983).
The preferred approach to attaining optimal calcium intake is through dietary
sources. Dairy products are the major contributors of dietary calcium because of their
high calcium content (e.g. approki"~alely 250-300 mg/8 oz of cow's milk) and frequency
of consumption. As used herein the term "milk" is unde~lood to refer to cow's milk,
and the term "dairy products" is understood to refer to food products derived from cow's
milk. However, many pe, ~ons, especially women, prefer to limit their intake of dairy
products for several r~asOn5: (a) they dislike the taste of milk/milk products; and/or (b)
they have a lactose i, ~ lerdnce; and/or (c) they per~eive that some dairy products are
too high in fat or protein and may lead to weight gain. Other good food sources of
calcium include some green vegetables (e.g. bloccoli, kale, turnip greens, Chinese
c~hb~ge), calcium-set tofu, some legumes, canned fish, seeds and nuts. Breads and
cereals, while relatively low in calcium, contribute siyl ,iricar,lly to calcium intake because
of their frequency of consumption. A number of calcium-fortified food products are
currently avaiiable, including fortified juices, fruit drinks, breads and cereals.
Consumption of these foods may be an addilional strategy by persons to achieve their
optimal calcium intake.
To maxi",i~e calcium absorption, food selection decisions should include
consideration of information on the bioavailabil;ty of the calcium contained in the food.
Bioavailability (absorption) of calcium from food depends on the food's total calcium
content and the presence of components which enhance or inhibit calcium absorption.
Bioavailability of minerals in food has been traditionally tested by the balance method,
which estimates absorption from the difference between ingested intake and fecaloutput. This approach works well for many nutrients where the difference betweenintake and excretion is large, but is less well suited for an element such as calcium
--3 -
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er,leri- ,g the digestive tract with its sec, e :lions. A decline in fractional absorption from
30% to 20% could have profound nul, ilional significance but would be difficult to detect
using the balance method. In conl,dsl, is~t-Fic methods e~li",dle absor~.lion directly
from the appearance of the ingested tracer in body fluids. Future clinical evaluations of
the bioavailabil;ly of calcium from the liquid nul, ilional product of the presenl invention
will use a state-of-the-art isotope tracer method.
Not all calcium salts are created equally. Calcium salts range from 9% elementalcalcium in calcium gluconate to 40% calcium in calcium carbol,dle. Bioavailability
depends on solubility. A new calcium delivery system, Calcium Citrate Malate (CCM)
claims to be approximately six-times the solubility of either calcium citrate or calcium
malate, both of which are themselves suL ~sldnlially more soluble than calcium
Cdl Lonale. Smith et al., "Calcium Absorption from a New Calcium Delivery System(CCM)" CALCIFIED TISSUE INTERNATIONAL, 41 :351-352 (1987) relates an
expe, i" ,enl in humans wherein calcium from CCM was absorL,ed s4"iricar,l1y better than
from either calcium carbonate or milk. 38.3% vs 29.6% and 29.4% respectively. WO91/19692 .I;,closes a process for making a met~ ' ~'e calcium citrate malate.
However, the United States Food and Drug Adllli, l;~ lion (FDA) has advised
that, in order for calcium-conl~i. l;. ,9 food i"y~ ed,e. IL~ in convenlional foods or calcium
sl~, ~len,ent products to be considered eligible to bear the authorized
calcium/osteoporosis health claim, they must meet the requi,~l"e"l~ in 101.14, which
include that they have been shown to the FDA's satisfaction to be safe and lawful under
the ~pplic- ~'e safety provisions of the act (56 FR at 60699). Safety and lawfulness can
be demon:jLI dted in a number of ways, including through a showing that a food is
generally recognized as a safe (GRAS), affirmed as GRAS by the FDA, listed in the food
additive regulations, or subject to a prior sanction. Of the 36 or more calcium-containing
ingredients idenliried by the agency as currently in use the FDA advised that only the
following 10 compounds had been demonsl, ~led to be safe and lawful for use in adietary supplement or as a nutrient supplement: calcium Cdl bonale, calcium citrate,
calcium glycerophosphate, calcium oxide, calcium pantothenate, calcium phosphate,
calcium pyrophosphate, calcium chloride, calcium lactate, and calcium sulfate (56 FR at
60691).
Table 2 summarizes the enhancement and i"hil,ilion factors associated with
calcium absorption.
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TABLE 2
FACTORS WHICH ENHANCE OR INHIBIT CALCIUM ABSORPTION
Inhibitors Enl.a.. cer~
Older age (~ 51) Younger age (11-24)
Vitamin D deric ncy Healthy vitamin D levels
Oxalic acid, fiber & phytates (only if Pregnancy & l~ct~tion
achlorhydria present)
Esl,ogen (natural & ,~:place",er,l
therapy)
Caffeine ~ Adequate protein intake
Presence of other nutrients in Ca+2 s~FF'e."ent Ca+2: P04 ratio of 1:1
Excess protein intake ~ 2 X RDA Specific disaccha, ides: fructose &
lactose
Specific organic acids:
Citric
Malic
Ascorbic
Calcium absor,ulion is directly affected by an individual's vitamin D status.
Vitamin D der: ~nl individuals absorb less calcium than individuals whose vitamin D
stores are adequate. Vitamin D m~GI_b~" . ~ enhance calcium absor~ lion. The major
metabolite 1,25-dihydroxyvitamin D, stimulates active transport of calcium in the small
intestine and colon. Deri.,;en~y of 1,25-dihydroxyvitamin D, caused by inadequate
dietary vitamin D, in~dequ~te exposure to sunlight, impaired activation of vitamin D, or
acquired ~si~lance to vitamin D, results in reduced calcium absor,ulion. In the absence
of 1,25-dihydroxyvitamin D, less than 10 per~enl of dietary calcium may be absorbed.
Vitamin D deficiency is associated with an increased risk of fractures. Elderly palienl~
are at particular risk for vitamin D d~:ri~,;en.,y because of insuffficient vitamin D intake
from their diet, impaired renal s~,llhesis of 1 ,25-dihydroxyvitamin D, and inadequate
sunlight exposure, which is normally the major stimulus for endogenous vitamin DS~l ILI ,esis. This is especially evident in homebound or institutionalized individuals.
Supplementation of vitamin D intake to provide 600-800 lU/day has been shown to
improve calcium balance and reduce fracture risk in these individuals. Sufficient vitamin
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D intake should be ensured for all individuals especi~"y the elderly who are at greater
risk for dcvelopment of a deficiency. Sources of vitamin D b~sides supFl-rller)la include
sunlight vitamin D-fortified liquid dairy products cod liver oil and fatty fish. Calcium and
vitamin D need not be taken together to be effective. FYcessive doses of vitamin D may
introduce risks such as h~l en -' llria and hyperc-'o :nia and should be avoided.
Anticonvulsant me- l ~lions may alter both vitamin D and bone mineral met~ho'i~ "
particularly in certain d;sorder:i in the institutionalized and in the elderly. Although
s~",pLu"ldlic skeletal .li;eAse is uncommon in noninstitutionalized s~lLi"y:, optimal
calcium intake is advised for persons using a"licon~/ulsants. "Optimal Calcium Intake"
JOURNAL OF THEAMERICAN M~EDICAL ASSOClATlON, 272(24): 1942-1948 (1994).
A number of other dietary factors can also affect calcium absorption. Dietary
fiber and phytate have been i",plic~led as illhiLililly substances. The binding of calcium
by dietary fiber incl eases with increasing pH. The onset of prec;~ ion of calcium
phytates occurs in the pH 4-6 range as in achlorl,ydria. At low gastric pH values (2-3)
phytate does not bind calcium and calcium binding by dietary fiber would be weak if at
all. Thus in normal individuals calcium would reach i"Lesli"al sites as soluble spe~ s.
Dependi"y on the concer,ll ~lions and binding ~ r,ylhs of various food ligands some of
the calcium will be absorbed at the i"l~ al sites while the remainder becomes bound
as insoluble fiber and phytate cor, F'~xes. Challlpaglle "Low Gastric Hyd,ùcl,'oric Acid
Secretion and Mineral Bioavailability" ADVANCES IN EXPERIMENTAL MEDICINE
AND BIOLOGY, 249:
173-184 (1989).
Simple sugars and organic acids also have an effect on bioavailability. Fructosein orange juice and apple juice prur"ùled positive calcium bioavailability from Calcium
Citrate Malate (CCM) which is a combination of CaC03 citric acid malic acid: 5:1:1
mol/mol/mol). The lactose in milk forms a soluble compound with calcium. Organicacids such as citric acid malic acid and ascorbic acid may also play a role in the
favorable absorption of calcium from CCM. Mehansho et al. "Calcium Bioavailabilily and
Iron-Calcium Interaction in Orange Juice" JOURNAL OF THEAMERICAN COLLEGE
OFNUTRITION, 8(1):61-68 (1989).
In addition it is known that high protein intakes specifically of sulfur conlai"i"y
amino acids increase urinary calcium excretion. Sulfuric acid radicals are believed to
decrease renal tubular resorption. However consumption of high phosphorLJs foods
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such as meat, can di~ ish this effect. Spencer et al., "Do Protein and Pl .osphorous
Cause Calcium Loss?", JOURNAL OF NUTRI TION, 1 18:657-660 (1988) .
For some individuals, calcium supplements may be the preferred way to obtain
optimal calcium intake. Although calcium s~p~!e "ents are available in many salts,
calcium calbon~le is usually recon""ended because it cGnl~i.,s more elemental calcium
per gram than any of the other salts. The di~il IL~yl dlion and ~issc'ution ul ,ara~ri:,lics
of commercial calcium car ondle pleparalions, which vary widely, may produce
important differences in calcium absor,~lion. Other problems with using large amounts
of calcium carbonate is that it can lead to con~lic,~lion and abdon,i"al distention. When
problems arise, calcium lactate or calcium citrate are advised. These sl ~hstitl'tions for
calcium carbonate are also i,-cl c~l~d for people with achlorhydria. A popular
commercially available calcium s~ !e.--ent is TUMS 500TM which is distributed bySmithKline Beecham, Pittsburgh, Pennsylvania, U.S.A. and is labeled as providing 500
mg of ele."enldl calcium (from calcium carbonale pertablet). I loJJ_vcr, the TUMS
500TM label does not illdicdle that this calcium sul,plen,el,l conldi"s any vitamin D.
U.S. 4,786,510 and U.S. 4,992,282 c lisc,lose the use of calcium citrate malate in
a beverage or dietary sucrF!e.,,ent ~lliried with iron, but do not di,c,lose the addition of
vitamin D to such a product. WO 92/19251 and WO 92/21355 ~lisulose the use of
calcium citrate malate in a low pH beverage, and suggests that vitamin D be added to
such a beverage along with oil flavors or v ~igl ,i. ,g oil. However; neither WO 92/19251
or WO 92/21355 disclc!se any other details about how to incorporate vitamin D3 into
such a beverage.
EP 0 486 425 A2 discloses a liquid oral nutritional formulation which contains
carbohydrates, protein, fat, fiber, calcium, and vitamin D, and has a pH of about 3.5 to
3.9. However, this p~ Ihlic~tion teaches that high amounts of micronutrients such as
calcium or magnesium may impair the palatability of the product, and should contain the
recommended daily allowance of these nutrients in about one liter or product. In an
example in the patent publication this product contains only about 570 mg of calcium per
liter and about 211 IU of vitamin D per liter. A commercially available product in
accordance with this patent publication is distributed by Sandoz Nutrition under the trade
name CITRISOURCE~ and is labeled as providing 570 mg of calcium and 210 IU of
vitamin D per liter. By way of comparison, prototypes of a beverage according to the
present invention contain about 1,408 mg of calcium per liter and about 338 IU of
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vitamin D3 per liter.
U.S. 4,737,375 teaches beverage concenl,dles and beverages having a pH of
2.5 to 6.5, preferably 3.0 to 4.5, which cor,lai"s calcium. The use of vitamin D3 in this
beverage is not ~~isclosed. This patent does not teach the use of calcium
glycerophosphale (which is used in prer~r,ed embodiments of the prt:senl invention, as
a calcium source. The ~cidu'~nts used in this prior art beverage are chosen frommixtures of citric acid, malic acid and phosphoric acid, and the weight ratio of total acids
to calcium is in the range of 4 to 7. The calcium level is 0.06% to 0.15%, pl~ bly
0.10% to 0.15% of the beverage, by weight. By way of cor"pari~on, prototypes of the
beverage of the present invention have a weight ratio of total acids to calcium of about
5.1.
Two commercially available beverages which are labeled as being p, uteuled by
U.S. 7,737,375 are: (1) Sunny Delight~ With Calcium which is distributed by Procter &
Gamble, Cillc;.lndli, Ohio 45202 U.S.A.; and (2) HAWAIIAN PUNCH~, DOUBLE C
which is distributed by Sundor Brands, Inc., Ci"c;l "-ali, Ohio 45202 U.S.A. Acco,d;. ,g to
the "Nutrition Facts" on the labels of these cor"",er-,;ally available products: (a) either
product contains vitamin D; (b) neither product conlai. ,s any fat; (c) a 240 mL (8 fluid
ounce) serving of Sunny Delight~ With Calcium provides 30% of the recommended
daily intake of calcium; (d) a 240 mL (8 fluid ounce) serving of HAWAIIAN PUNCH~,
DOUBLE C provides 15% of the recommended daily intake of calcium; and (e) and a
240 mL (8 fluid ounce) serving of each of these products provides 100% of the
recommended daily intake of vitamin C. Per the product labels, these percent daily
values are based on a 2,000 calorie diet. A review of the ingredient listings on the labels
of each of these products i".licdles that both of these beverages are aqueous solutions,
and that neither product conldi"s gum arabic. Samples of each of these products were
tested regarding their pH values: the pH value of the HAWAIIAN PUNCH~E3) DOUBLE C
was 3.91; and the pH value of the Sunny Delight~ With Calcium was 4.05.
GB 2 196 253 A discloses a beverage conld;l l;l l~ calcium and vitamin D. A
water soluble non-toxic calcium salt is used in a quantity sufficient to provide in the final
beverage a calcium ion content of from 1.0 x 1 o-2 to 40 x 1 o-2% wlw. The beverage may
contain up to 5 x 106 w/w of vitamin D. However, this published patent application does
not teach the use of a gum, su~ch as gum arabic or gum tragacanth, in such a beverage
to improve vitamin D3 stability.
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The NIH Consensus Statement recommended that the private sector play an
active role in promoting optimal calcium intake by dcvel~p:.,y and l"arl~eli"g a wide
variety of calcium-rich foods to meet the needs and tastes of a mulli~ll,ni~ population.
"Optimal Calcium Intake", IQURNAL OF THE AMERICAN MEDICAL ASSOCIATION.
272(24):1942-1948 (1994). Hence, there is provided in acco,dance with one aspect of
the present invention a low pH beverage fortified with calcium and vitamin D3 There is
provided in accor lance with anolher aspect of the invention a liquid beverage
concer,l, dle fG, liried with calcium and vitamin D3. There is provided in accordaoce with
yet another aspect of the invention a liquid beverage additive fortified with calcium and
vitamin D3.
SUMMARY OF THE INVENTION
Thus, in a first aspect, the invention cGr"priaes a beverage concer,l,al~
c~" ,~,riai"y.
a) a source of calcium; b) vitamin D; c) a vegei ''e oil; and d) a gum.
This concer,l, ~le may be in dry powdered form or it may be in liquid form, in which case
it further co",priàesa quantity of an aqueous solution, usually water or juice. Either
concentrated form can be reconstitute~/diluted with an aqueous solution to form the
desired, final liquid beverage, which forms a second aspect of the invention. Suitable
solutions include water, fruit juices and ve~el ''o juices, among others.
The source of calcium prt:~r~bly is calcium glycel uphospl)ale, but may also be
calcium citrate malate or calcium carbonate or another food grade calcium salt. The
gum may pr~r~rably be selected from gum arabic, gum tragacanth and xa"ll,an gum;whereas the vegelN~le oil may p,~ferdbly be selected from corn oil and partiallyhydrogenated soybean oil.
Rega,.lless of form (dry concenl~le, liquid concelllldle or liquid beverage) thecompositions may further contain supplemental ingredients, such as vitamin C, lactic
acid, an ~cid~ nt, a sweetener, a glucose polymer, potassium benzoate or a flavoring
agent. Iff desired, the beverage may be carbonated.
In another aspect, the invention provides a calcium supplement in solid form
con,pli~i"g calcium glycerophosphate, vitamin D and vitamin C. Preferably, the calcium
supplement in solid form comprises calcium glycerophosphaLe, vitamin D3, vegetable oil,
vitamin C, and a gum selected from the group consisli"g of gum arabic, gum tragacanth
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and xa~lll,an gum.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1-7 are repr~senl~ e of the methodology used in determining vitamin D3
levels; and
Figs. 8-11 are representative of the methodology used in dt:l~nllillilly vitamin C
levels.
DETAILED DESCRIPTION OF THE INVENTION
The levels, half lives and other chara~l~ri~lics and prupe, lies of vitamin D3,
calcium and vitamin C It~ d to herein and in the claims were determined, and in the
intel~,reL~lion of the claims are to be del~rlllilled, accoldi,lg to the methods set forth in
the Appendix A alldched to and made part of this speciric~lion.
SELECTION OF INGREDIENTS USED IN PRACTICING THE INVENTION
The present invention provides high levels of calcium and vitamin D in a
carbonated beverage, a noncarbol1ated beverage, a liquid beverage concerllldle, a
powdered beverage concerlll dle, a powdered beverage additive, beverages containing a
powdered beverage concerll, ~l~ or additive, or a calcium sl ~ 'ament. As used herein
and in the claims the terms "liquid nul,ilional product" and "beverage" are under:,lood to
be synonymous. As used herein and in the claims a "low pH beverage" is ulldel~lood to
refer to a beverage having a pH of less than 4.6. Trial batches of low calorie lemon lime,
orange, peach, and wild cherry flavored prototype carbonated beverages have beenmanufactured in accordance with the prt:senl invention. The prototype beverages were
manufactured by preparing a beverage concer,l, ~le, then blending the beverage
concenl,~le with treated water. The blends where then carbonated and filled intoslandard 12 ounce soda aluminum cans. (Soda aluminum cans are coated in accordance
with accepted industry standards to substantially reduce migration of aluminum into the
conl~nl~ of the can.)
Calcium Source. As used herein and in the claims the term "calcium" used alone
-10-
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refers to elemental calcium, the term "calcium salt" refers to a chemical composition
containing elemental calcium, and "calcium source" refers to calcium and/or a calcium
salt. The calcium salt used in pr~r~"ed embodi",elll~ of the pr~:sel)l invention is Calcium
Glyceruphosph~Le (CaGP) which is generally recognized as safe (GRAS) by the United
States Food and Drug Ad"~i"i. l~lion (FDA) (21 CFR 170.3). Another reason for
selecting CaGP is that, as already ~ clQsed above in the background section, it is one of
the ten calcium compounds ,~c,oy"i~ed by FDA as safe and lawful for use in a dietary
sl~FFl_n,ent or as a nutrient supplement for osteoporosis. However; any other sUjtr~le
calcium source, such as calcium citrate malate that would be soluble at a pH of about 3.5-
4.5 could be employed in the practice of the p,~serll invention.
Calcium glycerophosphdle (CaGP) can be described as a white, odorless, almost
~sl~ ss powder. Its scl ~' ' 'y in water increases in the presence of citric and lactic acids,
as stated in the Merck Index. The CaGP used in the trial bdlcl)es was FCC lll grade and
was produced by Dr. Paul Lohman GmbH, Emmerthal, Germany and is distributed by
Gailard Scl~ ,ger Industries, Inc., Carle Place, New York,11514, USA.
Another reason for selecting CaGP is its excellenl calcium bioavailability. Churella
et al., "REL ATIVE CALCIUM (CA) BIOAVAILABILITY OF CA SALTS USED IN INFANT
FORMUL AS", THE FASEB JOURNAL, 4(3):A788 (1990) reports a study which
determined the calcium bioav '-~:' 'y of four calcium salts. Rats were fed various diets
containing dirr~,~r,l calcium salts for three weeks. At the end of the study, the right femur
was removed and tested for calcium. As co",pared to a control, the relative calcium
bioavailability was as follows: Il ;~a'c ~m phosphdle 110%, calcium citrate 110% and
CaGP 106%. Furthermore, studies r~ ol Led by Hanning et al, "Efficacy of calciumglycerophosphaLe vs conventional mineral salts for total palt:r,ler~l nutrition in low-birth-
weight infants: a rarldo",i~ed clinical trial'~3", AMERICAN JOURNAL OF CLINICAL
NUTRITION, 54:903-908 (1991), and Draperet. al., "Calcium Glycerophosphale as a
Source of Calcium and Phosphorous in Total Par~nlcr~l Nutrition Solutions", JOURNAL
OF PARENTERAL AND ENTERAL NUTRITION, 15(2):176-180 (1991) showed in low
birth weight infants and piglets, respectively, that CaGP is as effective as calcium
gluconate as a source of calcium in total parenteral nutrition (TPN) solutions and could be
used to prevent under mineralized bones in low birth weight infants.
Yet another reason for selecting CaGP was its high solubility which f~cilit~tes a
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larger calcium intake per serving. A number of calcium salts were evaluated for their
functionality in the liquid nutritional product of the presenl invention: ~;c~ci~lm phosphale,
monocalcium phosphale, calcium chloride, tricalcium phosphdle, calcium citrate, calcium
carL,ondle, CaGP, and D-gluconic acid (hemicalcium salt). Aqueous solutions conl~.i.,i"g
500 mg of calcium per 237 mL (8 oz.) serving (2110 ppm) were plepdl~d and the pH was
~fijusted to pH 3.5 and pH 5Ø Results indicated that solubility of calcium salts varied
and only calcium Cdl bondle, calcium chlo~ ide, CaGP, and D-Gluconic acid, remained
soluble at pH 3.5 for at least one month. In this evaluation sol 1hility was del~""i"ed by a
visual examination. At pH 5.0 all samples formed crystals over time. The results of this
solubility study are presenled in Table 3.
TABLE 3
SOLUBILITY OF CALCIUM SOURCES
(Solutions at 500 mg calcium per 237 mL)
Salt At Time of Manufacture 1 MONTH
pH 3.5 pH 5.0 pH 3.5 pH50
Dicalcium insol~ ' 'e insoluble inso' ~' 'e insol~' !e
Phosphdle
Monocalcium insoluble insoluble insoluble insoluble
Phosphal~
Calcium soluble soluble soluble insc' ' !e
Chloride
Tricalcium inscM' !e illsQ' ~' !e j"s~l l' 'e insol ~' 'e
Phosphdle
Calcium insoluble insoluble insoll''o insoluble
Citrate
Calcium soluble partially soluble ills~l l' !e
Carbonate Soluble
CaGP soluble soluble soluble insoluble
D-Gluconic- soluble soluble soluble partially
Acid* Soluble
* Hemicalcium salt
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Experiments were repe~ted with calcium carbonate, CaGP, and calcium chloride
in a complete liquid nul,ilional product matrix, i.e., in conjunction with aspd,ldl"e, a flavor
system and vitamin C. The pH range ev~ ted was 3.54.5. At the lower end of the pH
range all calcium sources were soluble at time of manufacture. After one month it was
observed that as the pH increased, calcium Cdl bOndle and CaGP formed crystals, worse
in the case of calcium Cdl Londle. In addition, it appeared that the CaGP had a synergistic
effect with aspa, lar"e regarding sweetness. Calcium chloride was cor"~let ~ ly soluble
throughout the pH range but its bitter flavor made it Ul ,acce,~ lable for the liquid nutritional
product of the present invention a~pliG;tlion. Calcium lactate was evaluated in
s~ ~hsequent experiments. Although its sol ~hi'ity was excellent it provided a~l, i"ger,l and
mineral salt-type notes to the taste of the beverage that made it u"desi, ' 'e.
Still another reason for selecting CaGP is the fact that a beverage matrix
containing this calcium salt requires the addition of less acid to acl.i_vc a pH below 4Ø
Acidity is desired in the liquid nul, ilional product of the plesenl invention for several
reasons such as: to Illaillldi.l the calcium salt solubility, to cor, r!en,enl flavor, to control
".i obial growth, and to enhance the role of preservatives, specifically potassium
ben~ dle or sodium ben~odle. On the other hand, too much acidity can result in
increased lal ll ,ess and sourness that make the product u"desi, ' ' = from a sensory point
of view. When calcium salts are added to the liquid nutritional product of the pr~senl
invention, the solution resists chanyes in pH and more acid is needed to bring down the
pH than in commercially available sodas with no calcium fortification. Aqueous solutions
of various calcium salts were prepared to deliver 500 mg of elen,enlal calcium per 12 oz.
serving (1408 ppm) and the pH adjusted to pH 3.5 with citric acid. Till ' "e acidity was
determined by measuring the amount of 0.1N NaOH needed to raise the pH to 8.3 in a
409 sample co"'- ,i"g 1,409 mg/Kg of a calcium source. The results presented in
TABLE 4 indicate that, with the exception of calcium cl-loride, CaGP was the calcium salt
that had the lowest lill ' ~'e acidity. Till ' ' l~ acidity is an indication of the total acidity
of a beverage.
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W O96/31130 PCTrUS~ SC01
TABLE 4
TITRABABLE ACIDITY OF CALCIUM SOURCES
Calcium Source Tifratable acidity
mL of 0.1N NaOH
Calcium Chloride 0.7
CaGP 43.5
Calcium Lactate 47.1
Tricalcium Pl ,osphale 48.6
Calcium Citrate Malate 53.2
Calcium Citrate 57.5
Calcium Hydroxide 60.6
Calcium Carl,onale 61~4
Calcium glycerophosphate (CaGP) is created by the reaction of glycerophosphate,
a weak acid with pKf=6.1, with the strong base calcium hydroxide. GaGP binds calcium
with an approximate fu~malio~ con~ nl of 1.7. CaGP, when dissolved in water,
dissociates readily to provide "free" calcium ions and p, ulon~led glyu3rophosph~l~
species. Acid-base buffering by monoprulondled glycerophosphale is effective only
within the pH range from 4.1 to 8.1 (refer to the Hender~on I l~ssell ~al,k equation), and
thus, GP exhibits il ,siy"irlca"l buffering capacity at pH=3.6. On the other hand, anions,
such as malate, tartrate, pr~p.onale or succ;"~le, do provide buffer capacity at pH=3.6,
and accordi. Igly require more base or acid than GP for final adjustment of pH.
Yet another reason for selecting CaGP is the low aluminum content in
commercially available CaGP. It has been theorized that chronic use of calcium
supplements which have siyl ~iricanl aluminum conlenls may constitute unnecessary metal
exposure. Whiting, "Safety of Some Calcium Supplements Questioned", NUTRITION
REVIEWS. 52(3):95-97 (1994). The aluminum content of some calcium sources is
presented in TABLE 5.
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TABLE 5
ALUMINUM CONTENT OF CALCIUM SOURCES
Calcium Source A/uminum Content
in parts per mil/ion (ppm)
CaGP 4.55
Calcium Hydroxide 300400
CaCO3 (from fossil shell) 4,4002
CaCO3 (from Dolomite) 171-3152
' Values deler~ ed by analysis of commercially available compounds.
2 Values from Whiting article.
It has been sugge: ilecl that calcium citrate may play a role in enhal)~i"g aluminum
absorption from food, polenlially resulting in toxic serum and urinary aluminum levels.
Sakhaee et al., have successfully demon~l,alt:d however, that the provision of calcium
citrate alone without aluminum - conl~i. ,i"g drugs does not pose a risk of aluminum
toxicity in subjects with nor",ally functioning kidneys. Sakhee et al., "Calcium citrate
without aluminum a nlacids does not cause aluminum retention in p~lienls with fu"~ioni.,g
kidneys," BONEAND 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 understood to refer to vitamin D, cholecalciferol (D3),
ergocalciferol (D2) and its ki~logiG~Ily active metabolites and precursors such as, 1a, 25-
(OH)2 vitamin D; 25 OH vitamin D, its biological precursor; and 1a hydroxyvitamin D, and
analogues of the dihydroxy compound. These materials promote i"lesli"al absor~lion of
calcium, contribute to plasma calcium regulation by acting on the remodeling processes of
accretion and resorption and stimulate reabsorption of calcium by the kidney. While the
form of vitamin D3 used in the following exar,,Flos~ prototypes and experiments is
cholecalciferol, it is understood that any of the various forms of vitamin D may be used in
practicing the present invention, but vitamin D3 is preferred in embodiments which are
liquids.
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Dietary calcium and vitamin D are the natural mediators against bone loss.
Vitamin D acts directly on bone cells (osteoblasts, osteoclasts) to alter bone mass. It also
promotes gut uptake of calcium. Human skin activates pre-vitamin D molecules when
exposed to ultra violet irradiation. In the summer, 15 minutes exposure to sunlight is
sufficient to maintain adequate vitamin D levels. On the other hand, during winter, all day
exposure to sunlight will produce negligible conversion of vitamin D. The thinner skin
associated with aging is a less effective converter than the youthful skin.
The addition of vitamin D to the liquid nul,ilional product of the p,~senl invention
was difficult bec~use this is an oil soluble vitamin whereas both the beverage concenl, dle
and the beverage of the pr~:se"l i"~ lion are ~queous solutions. A number of po~ !e
methods to overcome the immiscibility of these two phases were ev~lu~ter~ The results
of these efforts are related below, and batch numbers are sequential throughout the
following studies.
There were two major obstacles to overcome regarding the i"Col ~ordLion of
vitamin D3 in the preser,l invention: (1) the initial prucessi"g loss of vitamin D3; and (2)
the stability of vitamin D3 over the shelf life of the product. To cor"pa, ~: the initial
processing loss and stability of vitamin D3 of each variable with successive batches, two
criteria were routinely measured: (1) % recovery of vitamin D3 at 0-time; and (2) half life
of vitamin D3 (t1~)
The % recovery of vitamin D3 of each batch was c~lcl ~l~tPd by dividing the 0-time
vitamin D3 result by the theoretical rO, liricdlion of each batch times 100%. (See Table 6).
As used herein "theoretical rO~ liricdlion" refers to amount of vitamin D3 added to the
product. As used herein "0-time" refers to the time of initial vitamin D3 analysis of the
product. In Table 6, "% Recovery" is the per~;enlage of theoretical fo, liricdlion of vitamin
D3 remaining in the product after initial plucessi,lg loss. Only batch 31 did not have the 0-
time vitamin D3 determined. Tl)ererurt:, a proj~ct~d result for this batch was extrapolated
from the negative exponential regression curve generated from the stability data.
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TABLE 6
O-TI_F VITAMI ~I n~ RF.~UI TCi VF ~SUS THFORFTICAI Ft~RTlFlcATlnN
BATCH 0-TIME THEORETICAL % RECOVERY
440 950 46.3%
- 2 405 950 42.6%
3 450 950 47.4%
Mean for batches 1-3 45.4%
4 249 635 39.1 %
283 635 44.6%
6 294 633 46.4%
Mean for batches 4-6 43.4%
7 371 483 76.8%
8 328 634 51.7%
9 308 618 49.8%
Mean for balches 7-9 59.4%
548 841 65.2%
11 696 844 82.5%
12 680 843 80.7%
13 691 844 81.9%
14 546 842 64.9%
649 845 76.8%
16 679 844 80.5%
17 681 844 80.7%
Mean for batches 10-17 76.7%
18 752 916 82.1%
19 678 915 74.1%
802 916 87.6%
(control batch)
21 784 917 85.5%
22 491 917 53.5%
23 796 916 86.9%
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W O96/31130 PCTrUS9''0~GOl
TABLE 6 (continued)
BATCH 0-TIME THEORETICAL % RECOVERY
24 7998 916 J 87.1%
Mean for balches 18-1978.2%
& 21-24
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)
576 823 70.0%
Mean for balcl ,es 25-28 66.6%
&30
31 786*** 840 93.6%
** No Data Available - Ex.,~pGI-'ed From the Exponential Rey~ession Curve
To better characterize the stability of vitamin D3 over time in all the bdlcl ,es the
Henri-Michaclic Mcnton ex~ oner,lial equation was e" p'oyed. The vitamin D3 results
(IU/KG) for each variable was plotted versus time (Day) and a ~ey,~:ssion curve was
fitted through the data using the follo.~-;"g equation:
lD] = [Do] e~kt
Where: [D] = Vitamin D3 conce"l,dlion (IU/KG) at time (t).
[Do] = Vitamin D3 concenll~lion (IU/KG) at 0-time.
e = Exponential
k = Rate conslanl (rate of ioss of vitamin D3 over time)
t = Time (days)
Stability was defined as the amount of time (days) that would be required for
the initial concentration of vitamin D3 to be reduced one half. This was termed half-life
(t"2). The more stable the vitamin D3 in a particular formulation the longer it would
take for the initial concentration to be reduced by one half. Rean dny;"g the previous
equation and making the appropridte sl lhstitutions~ the half-life of vitamin D3 in a
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W O96/31130 PCT~US96/04601
particular variable could be ex~,essed as:
t"2 = In 2/k
t"2 = Time (days) required for vitamin D3 to be reduced by one half of the
initial concenl,dlion.
In = Natural log.
k = First order rate constant (rate of loss of vitamin D3 over time):
The various bdlcl1es are described in the r "~J.~i,lg text. For convenience, thebatch numbers are sequential. In addition, the actual vitamin D3 data at each time
point for each respective variable are presenled in Tables 8, 10, 12, 13, 14, 16 and 17.
The correlation co~rri onls, initial vitamin D3 concer,l,~lion [Do]~ first order rate
consla"l:~ (k), and vitamin D3 half lives (t"2) are also pr~senled in Tables 8, 10, 12, 13,
14, 16 and 17 and should be referred to during the discussion.
A del '_d ~iscuscion of each variable will not be prt:senled since such a
presenl~lion would be quite lengthy. Rather an overview of various balcl ,es grouped
with respect to the main Vdl ~ '-'~ S that were studied will be ~iscusse~
a. Use of a Water Disper~ 'e Form of Vitamin D3
Early in the dcvelop",ent of the present invention an evaluation was made of a
water dispersable vitamin D3 spray dried on a dicalcium phosphale and gum acaciacarrier. The water dispersible vitamin D3 used in this evaluation was a DRY VITAMIN
D3 Type 100-DS pu,chased from Roche Vil~lll ,s and Fine Cher"icals, a division of
Hoffman-LaRoche, Inc., Nutley, New Jersey, U.S.A., which conl..i.,s vitamin D3
(chclec~lciferol USP-FCC), 1;~ phosph~le, gum acacia, coconut oil, BHT,
lactose, silicon dioxide, sodium ben~u~l~ and sorbic acid. It is a white powder and
contains 100,000 IU/g of vitamin D3.
Three batches were manufactured to evaluate the water di~per~ ' le form of
vitamin D3. Each batch consisted of an aqueous solution containing pc,lassium
benzoate, citric acid, sodium citrate, aspd~ lal"e, calcium glycerophosphate and the
water dispersible form of vitamin D3. The resultant product was not homogenized.The final pH of each batch is presented in Table 7. This pH difference, however, did
not seem to affect vitamin D3 recovery.
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WO96/31130 PCTrUS~6/O~C01
TABLE 7
BATCH pH
3.50
2 4.19
3 4.97
The initial prucessil ,g losses for bal~,hes 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 not homogeni~ed into the product matrix; and (b) there was no
emulsifier pr~se"l that would assist in maintaining the vitamin D3 in solution.
Therefore, the vitamin D3 was lost by the coating of the manufacturing equipment with
vitamin D3. The stability of Vitamin D3 in these three balcl-es was not ~cceFt~~'e over
the shelf life of the product. As shown in Table 8, one half of the initial vitamin D3 was
lost in approxil"dlely 12.6 days.
TABLE 8
VITAMIN D~ (IU/KG OF PRQDUCT) VERSUS DAYS
BATCH 1 2 3
Days1
o2 440 405 450
72 328 315 347
13 191 210 225
Corr. Coef. 0.955 0.968 0.965
[Dol 462 418 466
k 0.0636 0.0501 0.0529
t.,2 10.9 13.8 13.1
Average Half Life (t 1e) of Vitamin D3 for Batches 1-3 is 12.6 Days
Days after initial vitamin D3 testing. 0-time testing occurred 7 days after the
product was manufactured.
2 Results for batches 1-3 were corrected via control value on day 0 and day 7.
b. Use of Polysorbate 80 as an Emulsifier
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CA 02217264 1997-10-02
W O96131130 PCTrUS_~'01C01
A series of ex~.eri",ents were conducted using vitamin D3 in Polysorbate 80
manufactured to selected speciricdlions by Vildlllills Inc., Chicago, lllinois, U.S.A.
Polysorbate 80 is a water soluble, non-ionic emulsifier used for various ap~ ions in
the food industry. It is a polyoxyethylene derivative of sorbitan monooleate which
i"laracl:j with the oil and aqueous phases in an emulsion to form a barrier at the
interface that causes a reduction in Van der Waals forces and an improvement in
emulsion stability. It was e,~,uel,led that the use of Polysorbate 80 to incorporate the
vitamin D3 would improve its recovery and stability by causing di~ er~iGn of the oil
phase in the continuous ~queous phase.
The effect of Polysorbate 80 was evaluated in three ex,ueri,,,ental bdlcl-es of a
low pH beverage. Liquid beverage concer,l,dles were pr~:part:d as desc,ibed above,
i.e., adding to water sodium ben~oale (instead of polassium benzoate as a
preservative), citric acid, polassium citrate, aspa,ldl"e, calcium glycerophosphale, and
vitamin D3 in a premix cor,l~i.,;"g Polysorbate 80 and propylene glycol. The resultant
liquid beverage concer,l,dles were not homogenized and were diluted with five parts of
water before carbonation. The vitamin D3 fo, lificdlion level for each batch was 635
IU/KG of product. All bdlches contained vitamin C. The vdr ~les in bdlches 4-6 are
presented in Table 9. These Vdli~ s were added in an all~:lll,ul to improve vitamin C
stability, since it has been found that cysteine, when added in a carefully conl,~"ed
amount can overcome vitamin C deleriordlion in packaged beverages (U.S. Patent
3,958,017, May 18, 1976).
TABLE 9
BATCHVARIABLE
4Cysteine,1.5% of Vit. C
5 No Cysteine
6Cysteine + 250 PPM WPC
The overall mean % Recovery for batches 4-6 was comparable to the previous
- batches conldi"i"g the water dispersible form of vitamin D3. The mean % Recovery
was 43.4% (Table 6). However, as shown in Table 10, the stability of these bdlches
improved significantly. The half life of vitamin D3 in these batches ranged from 257
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W O96/31130 PCTrUS~'VlC01
days to 1,160 days. Cysteine addilion did not affect vitamin D3 recovery, but batch 6
with whey protein concenl~le (WPC) showed minimal loss of vitamin D3 during 60
days of shelf life.
In addilion, batch 6 also had slightly better initial vitamin D3 Recovery than
those bdlches in this series without protein. This suygesled that a more rugged
emulsion and some sort of matrix was needed as shown in Table 10. The use of WPCis not i"~ I;c-.led if the product of the invention is desired to be low in calories or free of
calories, but otherwise may be used in the practice of the invention. In an dll~ L to
make a low calorie or calorie free product, the use of mechal-ical means such ashomogeni~lion was inve~ !~d
TABLE 10
VITAMIN D3 (IU/KG QF PRODUCT) VERSUS DAYS
BATCH 4 5 6
Days'
0 249 283 294
7 226 272 282
243 243 281
261 239 279
Corr. Coef. 0.450 0.783 0.512
[Do] 236 275 288
k 0.0015 0.0027 0.0006
t"2 462 257 1160
Average Half Life (t1,2) of Vitamin D3 for Batches 4-6 is 626 Days.
' Days after initial vitamin D3 testing. 0-time testing occurred 2 days after the product
was manufactured.
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W O96/31130 PCTrUS9GI'~1C
C. Use of Homogeni~dlion
In the next series of studies, the vitamin D3/Poly~orL,dle premix was combined
with the aqueous phase and the blend was emulsified by passing it through a two-stage Gaulin-L-100 homogenizer at a given pressure. The purpose of this
homogeni~dlion step is to break up, or evenly disperse the oil phase into the aqueous
phase so that the particle size of the emulsion is Surr enlly small to retard
coalescence of the oil phase and prevent sepa~ dlion. A two-stage homogenization is
needed since the fine pa, licles formed during the first stage can clump. The second
stage, set at a lower pressure, is needed to break up the clumps, thereby making a
more stable emulsion.
Brominated vege: ~'e oil (BVO) and small quanlilies of gum arabic were added
to the vitamin D3/Polysorbate premix prior to homogehi~dlion. This was done to
increase the specific gravity of the oil phase and avoid phase separdlion or oiling-off
of the emulsion. BVO is used in the soda industry as a sl~ er for flavoring oils used
in fruit flavored beverages. BVO is a Food Additive (21, CFR 180.30) allowed in an
amount not greater than 15 ppm of the ri~ ,ished beverage.
A series of ex~,eri",ents were conducted to evaluate the effect of
homogeni~dlion on vitamin D3 recovery and stability. In these ex~ueri",ents liquid
beverage concenlldles were pr~pal~d as desc~iL,ed above with the exception of the
vitamin D3 addition. All the water soluble componenls were first dissolved in water and
a vitamin D3 emulsion, prepared separdl_ly was added at 1% of finished product
concenl,dlion and mixed thoroughly. The vitamin D3 emulsion was prepared by
cor"l.i"i. ,9 water vitamin D3 and one or more of the following ingredients: Brominated
Vegetable Oil (BVO) Polysorbate 80 Gum Arabic (GA), and corn oil followed by
homogeni~dlion using a two stage homogenizer. Two dirrt:r~nL sources of vitamin D3
were used: (a) an oil soluble vitamin premix where the vitamin D3 is dissolved in a
small amount of corn oil; and (b) a vitamin D3 premix where the vitamin D3 is dissolved
in Polysorbate 80 and propylene glycol (PG) (same as bdl~ l-es 4 through 6). One part
of the complete concentrate was then dissolved with five parts of water before
carbonation. The variables in batches 7-24 are presented in Table 11.
CA 02217264 1997-10-02
Wo 96/31130 PCTlu~J ~l~ ~C01
TABLE 11
Batch Variable
(ppm = ppm of product)
7 BVO, Vitamin D3 in Corn Oil, GA (0.14 ppm)
8 BVO, Corn oil, Vitamin D3 in PolyaorL~dte 80, GA (0.14 ppm)
9 BVO, Corn Oil, Vitamin D3 in Polysorbate 80
BVO, Vit D3 in Corn Oil, Polysorbate 80 (0.07 ppm), GA (0.14 ppm)
11 BVO, Vit D3 in Com Oil, Pol~/aorlJdL~ 80 (0.035 ppm), GA (0.14 ppm)
12 BVO, Vit D3 in Corn Oil, Poly:,o,L.ale 80 (0.035 ppm), PG (0.15 ppm), GA
(0.14 ppm)
13 BVO, Vit D3 in Corn Oil, Polyao~L~dl~3 80 (0.07 ppm), GA (0.14 ppm)
14 BVO, Vit D3 in Corn Oil, Polyaort~ale 80 (0.07 ppm), PG (0.30 ppm)
BVO, Vit D3 in Corn Oil, PolyaorL~aLe 80 (0.035 ppm)
16 BVO, Vit D3 in Corn Oil, Poly:,o,l,.lle 80 (0.035 ppm), PG (0.15 ppm)
17 BVO, Vit D3 in Com Oil, Pol~aorl~al~a 80 (0.07 ppm)
18 Same as 11
19 Same as 12
Same as 13
21 Same as 15
22 Same as 16
23 Same as 17
24 BVO, Vit D3 in Corn Oil, Polyao~l~ale 80 (0.07 ppm), Fructose (42,000
ppm)
The gum arabic used in all batches was Nutriloid Gum Arabic from Tic Gums,
Inc. When extra Polysorbate 80 was added to the batches, the percent addition refers
to percent of oil in the batch. Batches contain either 3% or 6% extra Polysorbate 80
added. The 20% and 40% refer to co~ nb;"~lions of Polysorbate 80 and Propylene
glycol where the Polysorbate 80 content is 3% and 6%. Fructose was added to batch
number 24 to see if it would extend the shelf-life of the product which is limited by the
degradation of aspartame. In general, the fructose and the various levels of
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CA 022l7264 l997-l0-02
W O96/31130 PCTrUS~ 1C01
POIy~Or~dLe 80 did not affect the vitamin D3 recovery as the homogeni~dlion step did.
The initial vitamin D3 Recovery (mean = 59.4%) and the mean half-life value
(150 days) for batches 7-9, as prt:sef lled in vitamin D3 Recovery Table 6 and Table 12,
in-~icat~sl that with few excepLions the homogeni~dLion step significantly improved the
initial recovery and stability of vitamin D3 versus previous dLLe,n~L~.
TABLE 12
VITAMIN D3 (IU/KG OF PRODUCT) VERSUS DAYS
BATCH 7 8 9
Days'
0 371 328 308
7 354 372 346
26 217 235 224
189 284 236
Corr. Coef. 0.816 0.224 0.506
[Do] 349 324 309
k 0.0098 0.0030 0.0047
tv2 71 231 147
Average Half Life (t"2) of Vitamin D3 for Ldl~ l.es 7-9 is 150 days.
Days after initial vitamin D3 testing. 0-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 for these batches d, ~r"dLically improved to 76.7%
versus all previous batches (Table 6). The overall vitamin D3 stability (mean half-life =
68.6 days) for batches 10-17 as presented in Table 13, was not as good as batches 7-
9 (150 days) but was superior in cor"pari~on to batches 1-3(12.6 days).
In orderto confirm the initial vitamin D3 Recovery and stability of batches 10-17
duplicate batches were made (see bdLcl1es 18-19 and 21-24 in Tables 6 and 14). The
CA 02217264 1997-10-02
W O96/31130 PCTÇUS~ SC01
initial vitamin D3 Recovery for balclles 18-19 and 21-24 (mean = 78.2%) corroborated
previous recoveries for batches 10-17. Furthermore the vitamin D3 stability of bal. hes
18-19 and 21-24 (mean half-life = 76.7 days) was cor"pal~ble to their respectiveduplicate batches (68.6 days).
-26-
CA 02217264 1997-10-02
W O96/31130 PCTrUS~'OlC01
0 0
OD ~ ~ ~ 0 ~ O ~
CD ~ ~ ~ ~ O U~ O 1--
c~ 0 r-- OD ~o ~~ ~ ~ ~.
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CA 02217264 1997-10-02
W O96/31130 PCT~US9''01C01
co ~ ~> o "~ c~ O a~
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- 28 -
CA 02217264 1997-10-02
W O 96/31130 PCT~USS~'~1C01
Although the shelf life data for batches 10-17 and 18-24 sho~.~d a loss of
vitamin D3 as a function time, no siy"iricdnl amount of de_~ dddlion product could be
analytically d~lected. Therefore, the main ",echar,i.~", for loss was assumed to be
physical ",4,dlion of vitamin D3 to the walls of the container, and/or rapid oxidalion of
vitamin D3 and/or isomerization of vitamin D3 to 5,6-trans-vitamin D3. Further studies
focused on incit:asi"g the emulsion stability to prevent the l"i~,dlion of the
hydrophobic vitamin D3 to the container walls.
d. Use of Gum(s) as an Emulsion Slabili~er
The use of gum arabic and gum tragacanth as emulsifying agents for flavor oils
in soft drinks is welbo ~ ~' ' ,ed in the soft drink industry. Melillo, "Physical Factors
Governing the Sl~ lion of Cloudy Beverages", FOOD PRODUCTS
DEVELOPMENT, June,1977, pp. 108-110. While only gum arabic was used in the
ex,ueri",e"ls, exdl", 'es and prototypes disclosed herein, it is u"der:,tood that one
skilled in the art could sl ~hstitute appl upridle amounts of gum tragacanth, xdnll Idl I gum
or any other apprupridle gum into the products of the pr~senl invention, or thatmixtures of gums may be used in the practice of the preser,l invention.
Gum tragacanth is the dried, gummy exudation obtained from Astragalus
gummifer or other Asiatic spe~ ~ s of Astralagus. Tragacanth swells rapidly in either
cold or hot water to a viscous colloic'-' sol or semi-gel. The mo'ecl ll:3r weight of the
gum is on the order of 840,000 and the molecl~4s are elongated (4500A by 19A) which
accounts for its high viscosity. Tragacanth gum is c~lllF ' Ie with other plant
hydrocoll~i~'c as well as carbohydrates, most prolei.ls, and fats. Viscosity is most
stable at pH 4 to 8 with a very good stability down to pH 2.
Xanthan gum is an exocellular heteropolysaccharide produced by a distinct
fermentation process. The bacterium xanthomonas campestris generates the gum on
specific organelles at the cell surface by a complex enzymatic process. The molecl ll~r
weight for xanthan gum is about two million.
Gum arabic, also known as gum acacia, is the dried, gummy exudate from the
stems or branches of Acacia senegal or of related species of Acacia. The most
unusual property of gum arabic among the natural gums is its extreme and true
solubility in cold or hot water. Gum arabic is a complex calcium, magnesium, and
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W O96/31130 PCTrUS~6/01G01
potassium salt of arabic acid. It has a main backbone chain of (1 , 3) - linked D-
g-'~cl~pyranose units, some of which are sllhstitllt~d at the C-6 posilion with various
side chains. The side chains consist of D-g-'nctopyranose, D-glucuronic acid and L-
arabofuranose with addilional side chains on the D-galactopyranose of L-
rhamnopyranose. The mo' eol ll~r weight is on the order of 250,000.
Gum Arabic is effective in ~ g emulsions and illhiLilillg co-'escence or
phase separation by two mechanisms: (a) increasing the viscosity of the continuous
(~queous) phase; and, (b) for",i"g strong films around the oil ~llop!_' . A small amount
of protein is presenL in the gum arabic as a part of the structure.
A series of experiments were conducted to evaluate various types of gum
arabic as the emulsifier system in the vitamin D3 emulsion. Although gum arabic had
been evaluated in previous experiments, the usage rate was too low (0.14 ppm) tohave a significant effect. It has been reported that the p,uL~ ceous component is
responsible for gum arabic's emulsifying and stabilizing prope, Lies. The vdl ~"'es in
b~Lches 25-30 are plt:sehLed in Table 15.
TABLE 15
Batch Variable
Gellan Gum, Kelco Products, 100 ppm (in beverage)
26 Gum Arabic, Tic Bev 202, Tic Gums Inc., 2000 ppm (in beverage)
27 Gum Arabic EMULGUM, Colloids Naturels Inc., 500 ppm (in
beverage)
28 Gum Arabic Nutriloid, Tic Gums Inc., 2000 ppm (in beverage)
29 Control, Same as Batches 13 and 20
Gum Arabic SPRAY BE, Colloid Naturels, Inc., 500 ppm (in
beverage)
Batches were prepared to evaluate the stability of various vitamin D3 emulsions
in finished beverages. The individual emulsions, prepared separately, were added to
beverage concentrates in amounts to yield 1 % by weight in the finished beverages.
The emulsions themselves contained 1-20% by weight of the appropriate gums whichwere first hydrated in aqueous solutions for about two hours at 60~C. (See Table 15
for gums and quantities) The hydrated gum solutions were cooled to 37.8~C or less
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CA 02217264 1997-10-02
W O96131130 PCT/US9C/O~C01
before the needed amounts of vitamin D3 were added. The type of vitamin D3 used
was liquid vitamin D3 in corn oil oblai"ed from Roche Vildmi, ,s and Fine Chemicals, a
division of I lorr",an-LaRoche Inc., Nutley, New Jersey, U.S.A. The pH of the
emulsions which contained gum arabic were lowered to pH 4.0 and sodium benzoate
was added to preserve the emulsions for e,clended use. The emulsions were then
homogeni~ed twice using a two-stage homo~eni~er at 1,500/600 PSI and 3,000/1,000PSI, respectively. For example, batch 27 contained 50 grams of EMULGUM gum
arabic hydrated in 950 grams of water, and upon cooling 77.2 milligrams of liquid
vitamin D3 in corn oil was ':lended into the gum solution in an amount giving a
theoretical fo~ liricdlion of about 825 IU/Kg of ri",shed beverage. The emulsion was
preserved by adding 0.3 9 of sodium benzoate and the pH was lowered to 4.0 by
adding 1.08 grams of citric acid.
The pe, r..r",ance of the different gums used, as indicated by initial vitamin D3
recovery and stability over shelf-life varied (Tables 6 and 16, respectively).
EMULGUM (batch 27) at 500 ppm concer,l~ dlion gave the best results followed by
SPRAY BE, both from Colloids Naturels, Inc.
In generaHt can be said that siy~iril,d~l improver,~enl~ in vitamin D3 stabilitywere observed initially and during shelf-life. The most significant improvement was the
stability of vitamin D3 over the shelf life of the product. The average half-life of vitamin
D3 for these bdlches was 180 days. It appears that at sufficient concentration, gum
arabic can coat the oil dl~Fl~t~ con Idil lil Ig the vitamin D3 to form an emulsion that can
be further st7hi~i~Pd by homogeni~dlion using a two-stage homogenizer.
This series of experiments demon:,l, dl~d that gum arabic could be substituted
for Polysorbate 80 to minimize initial processi"g loss and improve shelf life stability of
vitamin D3.
CA 02217264 1997-10-02
W O96/31130 PCTrUS9~'OlC01
,~n
o
~ ~ ~ ~ co C~~ ~ ~~ O 0
U~ U~ ~ ct~ C~o U~ o ~ --
o~
~ ~Dm ~
u~ o ~ o ~ ~ a~
CD ~ C~ C'' 1'- 0 C'' ~ 0
~ G ~
llJ C ~ ~,
~ ~ ~ ~~ ~ ~ ~ 8 -
o
."
~~ ~,.
C~ c~ ~ c~l C~ ~1 OD ~~ C~ g ~ '-- ~
z ~ ~ 0 N ~)
~ U~ C~ ~ C~l ~ ~O ~ O
~ > 'C-
> o a
a) ~ ~ ~
g~ C
~ ~ ~ ~ ~ ~ C~l O C~ O
I ~ I
~ ~ ~ C~J o ~ ~ C~l o C~ C
CA 02217264 1997-10-02
W O96/31130 PCTrUS3~'01G01
BATCH 31
e. Use of Commercially Manufactured Vitamin D3 Emulsion
In order to evaluate if a sl lit~ ~le vitamin D3 emulsion could be manufactured on
a larger scale which would support commercialization of a product accord" ,g to the
invention a decision was made to have the vitamin D3 emulsion manufactured by anoutside contractor. Taslt:r"aker Inc. of Cincinnati Ohio U.S.A. which is a providerof
flavoring products provided as a special order a vitamin D3 emulsion containing water
gum arabic partially h~dluger,dled soybean oil citric acid sodium benzoate and
vitamin D3. By actual analysis this cor"r"er- ;ally manufactured vitamin D3 emulsion
contains per 10 Kg: (a) about 9.52 Kg of water; (b) about 0.35 Kg of gum arabic; (c)
about 0.10 Kg of partially hydlugendled soybean oil; (d) about 0.02 Kg of citric acid; (e)
about 0.01 Kg of sodium benzoate; and (fl and at least about 787 000 IU of vitamin D3.
Tastemaker considers the manufacturing procedure it used to be prù~ - ie~a,y to it and
did not make that i- ,rur, . .alion available. While the CGI - -- ~ ,er~ ;ally manufactured
emu!sjon Gon!~ ed partia!!y hyd!--gPn~!Hd soybean oi! and the se!.f-manu.factur~d
emulsion conldil ,ed corn oil (see desc~ i~lion of bal- hes 25-30) it is understood that the
invention may be practiced using any sl~ e veget~le oil. Batches of which batch
31 is typical were manufactured as desc.iLled in previous e~,eri...er.l:,. The
commercially.manufactured vitamin D3 emulsion was added to the liquid beverage
concer~l~dle in an amount to equal 1% by weight of the finished beverage. The
beverage concer.l,~le was then added to water at a ratio of 1:5 and carbonated.
The initial vitamin D3 loss for batch 31 was minimal (94.1% recovery) which had
surpassed all batches to date. Furthermore the vitamin D3 stability of this batch was
superior to all previous balches. As presented in Table 17 the half life of vitamin D3
was 1 390 days.
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W O96/31130 PCTrUS9''0~G01
TABLE 17
VITAMIN D3 (IU/KG OF PRODUCT) VERSUS DAYS
BATCH 31
Days'
0 Not Tested
23 763
52 793
87 742
Corr. Coef. 0.218
[Do] 786
k 0.0005
t"2 1,390 days
Days after product manufacture, with day 0 being the day on which the
product was manufactured.
Acidulants. Acids are commonly used in food and beverages to impart specific
tart or sour tastes and to function as preservatives. A combi.-dlion of citric and lactic
acids are used in the liquid nul, ilional product of the pr~se"L invention. Citric acid is
the most widely used acid in fruit beverages in part because it blends well with these
flavors. It is commercially manufactured by fermenl~lion or by synthesis; either may
be used in the practice of the present invention. When using fermented lactic acid, a
purified form that is free of sugar rcsidues is recommended due to its cleaner taste and
clearer appearance. Food grade lactic acid is available in aqueous and crystalline
forms.
Sweetener. The sweetener used in the prototype beverages described below
is aspa, l~" ,e, but other artificial or natural sweeteners can be used in the practice of
the pr~senl invention. Artificial sweeteners that may be employed include saccharin,
acesulfame-K and the like. Natural sweeteners that may be employed include
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W O96/31130 PCTrUS9''01G01
sucrose fructose high fructose corn syrup gl-lcQse sugaralcohols dextrose
lode,~l- il ls maltose lactose and the like but other carbohydrates can be used if
less sweetness is desired. Mixtures of natural sweeteners or artificial swectcners or
natural and a, liri~ ial sw~:etener:j can be used also.
The amount of the l~weetaner effective in a product according to any aspect of
the present invention depends upon the particular sweetener used and the sweetness
i"lensily desired. In deter",;.,i"g the amount of sweetener any sugar or other
sJ~ ~ tener pr~senl in the flavor Gor"ponenl or product matrix should also be taken into
consiclerdlion .
Studies have shown that the efficiency of calcium absGrl,lion can be enhanced
two-five fold by oral admi";~l,dlion of glucose polymer both in patents with i"lt:sli"al
calcium malabsor~lion and in normal subjects. Kelley et al. "Effect of Meal
Co",posilion on Calcium Absor,ulion: Enhancing Effect of Carbohydrate Polyer'
GASTROENTEROLOGY. 87:596-600 (1984).
In another study using the triple-lumen intestinal perfusion techn:~l le glucosepolymer increased net calcium absor,ulion fourf ~old. Bei et al. "~''ucose Polymer
Increases and Equal Calcium Magnesium and Zinc Abso,l,lion in Humans"
AMERICAN JOURNAL CLINICAL NUTRITION. 44:244-227 (1986).
It is understood that a person of skill in the art may make a product in
accordal1ce with the invention corlldil lil l9 9l~ ~cose polymers or glucose.
Flavor. As used herein the term "flavor" includes both natural and artificial
flavors. The particular amount of the flavor col"ponent effective for i",pal li"g flavor
characteristics to the beverage of the present invention can depend upon the flavor(s)
selected the flavor i",pression desired and the form of the flavor component. The
amount of flavor employed in a product according to any aspect of the present
invention is within the skill of one in the art and depends on the flavor i"lensily desired.
Preservatives. Most microbial spoilage of low pH beverages is caused by
aciduric and acidophilic organisms like certain vari~lies of yeasts and molds. For this
reason preservatives with anti-microbial activity such as benzoic and sorbic acids are
added to soft drinks. Usage levels of these acids or their salts range from 0.025 to
0.050 percent depencli"g on the nutritive subsldnces present and the pH of the
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finished beverage. The a"li",ic,ubial activity of these preservatives has been shown to
be largely pH dependent. They are least effective under neutrai condilions but their
activity increases considerdbly with decreasing pH. For exa" ple, by reducing the pH
value from 4.5 to 3.0, the preservative effect of benzoic acid is i"c,~ased by nearly
three times. Only beverages at low pH receive the full benefit from the addilion of
preservatives. Woodruf et al., BEVERAGES: CARBONATED AND
NONCARBONATED, The AVI Publishing Company, Inc., 1974, pgs. 143-146. As with
most foods, the successful preservation of low pH beverages is dependenl on
controlling contamination of ingredients, prucessi"g equipment, and conlc.;"er:i by
poler,lial spoilage oryanisrlls~ Splittstoesser in FOOD AND BEVERAGE MYCOLOGY,
edited by Beuchatt, pulJlished by Van N~_',dnd Re;.,h~'d, 1987, pgs. 120-122.
Carbonation. The amount of carbon dioxide in a beverage accordi"g to the
present invention depends upon the particular flavor system used and the amount of
carbonation desired. Usually, carbonaled beverages of the pr~senl invention contai
from 1.0 to 4.5 volumes of carbon dioxide. P,~re"~d cd,l,onal~d beverages contain
from 2 to 3.5 volumes of carbon dioxide. The beverages of the pr~ser,l invention can
be prepar~d by ~landard beverage formulation tecl ",, les. To make a carbonated
beverage carbon dioxide can be introduced either into the water mixed with the
beverage syrup or into the drinkable diluted beverage to achieve carbonation. Itshould be understood, however, that ca,bonaled beverage manufacturing techniques,
when appropriately modified, are also app'i--~lE to noncarbonated beverages.
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EMBODIMENTS OF THE INVENTION
Tables 18-21 plesenl bills of materials for manufacturing prototypes of low pH
beverages ru~ liried with calcium and vitamin D3 in acco,dance with some aspects of the
invention.
TABLE 18
Bill of Malerials for Wild Cherry Fld~rGr.:d Bever~e
(For 1000 KG of Beverage)
INGREDIENT AMOUNT. KG
Treated Water' (for beverage cGncer,l,ale) 137.82
rol~-ss Mm Ben~oale 0.300
Sodium Citrate (dihydrate) 0.550
Citric Acid (anhydrous) 3.720
Lactic Acid (88%) 3.951
Aspa, l~",e 0.500
Calcium Glycerophosphal~: 8.331
vVild Cherry Color 0.000630
FD&C Red #40 0.0003465
FD&C Yellow #6 0.0002835
Natural & Artificial Wild Cherry Flavor 1.200
Ascorbic Acid 0.300
Vitamin D3 Emulsion2 10.000
Treated Water' (for final blend) 833.33
"treated water" has had the cl-lori"e and alkalinity ~ sted to levels cGr"r"or,ly
used in the soft drink industry.
2 This emulsion is described above with regards to batch 31.
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TABLE 19
Bill of ~ rials for Orange Fl..~rore.~ ~c~r~a
(For 1000 KG of Cc~er ~e)
INGREDIENT AMOUNT KG
Treated Water' (for beverage concentrate) 137.62
Boldssium Ben udle 0.300
Sodium Citrate (dihydrate) 0.550
Citric Acid (anhydrous) 3.720
Lactic Acid (88%) 3.951
Aspa"dr"e 0.500
Calcium Glyceruphosphal~ 8.331
Orange Color 0.0001875
FD&C Yellow # 6 0.00140625
FD&C Red # 40 0.00046875
Natural and Artificial Orange Flavor 1.400
Ascorbic Acid 0.300
Vitamin D3 Emulsion2 10.000
Treated Water' (for final blend) 833.33
"treated water" had had the cl.!ari"e and ~ rljusted to levels commonly
used in the soft drink industry.
2 This emulsion is desc, ibed above with l~:gar~ls to batch 31.
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TABLE 20
Bill of Mal~rials For Peach Fld~or~l Cever~2
(For 1000 KG of Geverdye)
INGREDIENT AMOUNT. KG
Treated Water' (for beverage concenl, dlè) 137.42
rOtaCSi~ l~ Ben~Gdle 0.300
Sodium Citrate (dihydrate) 0.550
Citric Acid (anhydrous) 3.720
Lactic Acid (88%) 3.951
Aspa, ld" ,e 0.500
Calcium Glycerophosphdle 8.331
Mohawk Casing Color 0.001250
FD&C Yellow # 6 0.0008125
FD&C Red # 40 0.0004375
Natural and Artificial Peach Flavor 1.600
Ascorbic Acid 0.300
Vitamin D3 Emulsion2 10.000
Treated Water' (for final blend) 833.33
"treated water" has had the chlori"e and alkalinity adjusted to levels commonly
used in the soft drink industry.
2 This emulsion is des~,iL,ed above with legalds to batch 31.
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WO96/31130 PCTrUS9~'ClC01
TABLE 21
Bill of Mdl~rials For Lemon Lime Flavored Ceve...~e
(For 1000 KG of ~ve.~.~e)
INGREDIENT AMOUNT. KG
Treated Water' (for beverage conce, 11l dla) 138.02
Bot~-cci~ Irn Benzoate 0.300
Sodium Citrate (dihydrate) 0.550
Citric Acid (anhydrous) 3.720
Lactic Acid (88%) 3.951
Aspartame 0.500
Calcium Glycerophosphate 8.331
Lemon Lime Color 0.000630
FD&C Yellow # 5 0.0005796
FD&C Green # 3 0.0000504
Natural and Artificial Lemon Lime Flavor . 1.000
Ascorbic Acid 0.300
Vitamin D3 Emulsion2 10.000
Treated Water' (for final blend) 833.33
"treated water" has had the chlorine and alkalinity ~ljnsted to levels commonly
used in the soft drink industry.
2 This emulsion is des~,iL,ed above with l~ald:, to batch 31.
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EXAMPLE 1
PREPARATION OF LIQUID BEVERAGE CONCENTRATE
The concel,l,dled mixture of i~y~dienl~ that make up the beverage is rer~r,t:d
to as the beverage concer,l,dle. The liquid beverage concenl~le cor"~lises at least
water a source of calcium vitamin D3 gum arabic and vegetable oil. rl~:ferdbly the
beverage concenl,dle also cor",~rises vitamin C. If desired the beverage concentrate
may also comprise: an acidulant preservative(s) andlor flavoring agent(s) and/or acid
stable co!ori"g agent(s). Prototypes of the beverage of the present invention have a
weight ratio of total acids to calcium of about 5.1. Prototype beverages of the present
invention contained vitamin D3 at levels of about 1.45 x 10~ to about 1.75 x 10~% wlw
and calcium at levels of about 1.46 x 10-' to about 1.47 x 10-' wlw.
In this example the liquid beverage concel,l,dle is pr~part:d in a single vessel at
ar" -ienl temperature by dissolving the ingredients in water using a ~ sndi"y tank
equipped with vigorous ayildliol, capability. A specihc order of addilion shown in
Table 22 is followed to aid in disper~i"g the ingredients in an t rr,~ ient manner. Each
ingredient should be completely dissolved before the next i"yl~d 3.,lis added.
TABLE 22
1. Water
2. rolassium Benzoate
3. Sodium Citrate
4. Citric Acid
5. Lactic Acid
6. Aspall~",e
7. Calcium Glycerophosph~l~
8. Acid Stable Coloring Agent(s)
9. Natural and Artificial Flavor(s) Agent(s)
10. Ascorbic Acid
11. Vitamin D3 Emulsion (vitamin D3 + gum arabic)
In commercial beverage manufacturing it is common for beverage
concenl,~les to be prepared a day or more (often weeks or months) in advance of
blending and filling conl~i"ers with the final product. For this reason the vitamin
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CA 022l7264 l997-l0-02
WO96/31130 . PCTrUS~'01C01
components may be added to the liquid beverage concenlldlt: just prior to blending
with water to complete the beverage in order to prevent unnecessary long term
exposure to air.
EXAIVIPLE 2
PREPARATION OF LIQUID BEVERAGE CONCENTRATE
Variations to the beverage concenl,dle manufacturing procedure des.;, iL,ed in
EXAMPLE 1 can be made if available mixing vessel sizes are limited and no singlemixing vessel is able to contain the required volume of beverage concenl, dL~e.
Beverages accordi.~g to the prese, ll invention have been manufactured by preparing a
plurality of beverage concel ,l, ale component slurries which were thereafter combined
by pumping each beverage conce"l, dle componenl slurry to a larger sized tank. The
water was divided equally between five clirrerenL beverage conce"l, dl~ component
slurries all of which were cor,~ld,llly ~git~t~l A first beverage concel ,l~dle component
slurry was made by first adding potassium ben,oale and then sodium citrate to the
water. A second beverage concenl, dle cor"ponenl slurry was made by adding to the
water in the following order: (a) citric acid; (b) lactic acid: (c) aspal ldr"e; (d) calcium
glycerophosphale. A third beverage concenl~ dle cor"ponenl slurry was made by
adding the acid stable ool~ori"g agent(s) and then the flavoring agent(s) to the water. A
fourth beverage concenll dle component slurry was made by adding the ascorbic acid
to the water. A fifth beverage concenl, ale cor"ponenl slurry was made by adding the
vitamin D3 emulsion to the water. The beverage concenl,dle cor"ponent slurries are
transferred to a single larger sized vessel in the order in which they have beendescribed. The resultant blend (the beverage concenl, dle) in the larger sized vessel
was vigorously agitated for not longer than about two minutes to homogeneously blend
the beverage concenl,dlt: component slurries together. A liquid beverage concenl,dle
in accordance with the invention should have a pH of 2.84.6 preferably 3.1-3.8. The
pH of the prototype beverage concenl,dtes typically ranges from 3.1-3.8. If necessary
additional lactic acid is used to adjust the pH of the beverage concentrate to this range.
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EXAMPLE 3
PREPARATION OF CARBONATED BEVERAGE
Deareation and cooling inc, t:ases the beverage's carbonation efficiency and
stability because the solubility of carbon dioxide in water is directly propo, lional to
carbon dioxide pressure and inversely propo, lional to ter"pe, dLure. The extent of
carbonation is expressed in terms of carbon dioxide gas volumes. The number of
volumes can be deler",i"ed by cor"pari"g sample readings with carbon dioxide
temperature/pressure r~ldLionship charts. Since pressure gauges measure the sum of
pressures from all gases the presence of air in the carbor,aLed mix can cause errors in
CO2 volume determination unless co~rections are made. A Zahm & Nagel air tester
makes it possible to easily measure the pressure and air content of a sample. Tomake such a test the sample conLai"er is pierced :~'ICJ:;, ,9 head space gases to be
~ ~le3scd into a buret filled with 10-20% sodium or potassium hydroxide. The carbon
dioxide is absorbed by the basic solution leaving only air inside the burette. The total
pressure reading is then corrected for the amount of air ~ ser,L in the burette
resulting in the corrected CO2 pressure. The gas volumes of the sample are then
deLerl"i.,ed using the corrected pressure.
A beverage in acco,dancê with the invention may be carbonated by either
blending the beverage concenL, dLe with cdrlJondLed water or blending the beverage
concenL, dLe with water followed by carbonation of the blend. The prototype beverages
were manufactured using a 5 to 1 ratio of beverage concenL, dLe manufactured
according to Example 2 to non-carbonated water. CarbondLion levels in the ri"ished
beverage may range from about 1.04.5 volumes of CO2, dependi, ,9 on flavor or
desired sensory attributes. The product is then packaged and sealed in aluminum
cans or tinted glass bottles. During the production of the prototype beverages
separate in-stream lines of beverage concenL, dLe and water were cor, Ibi"ed in the
proper ratio by a continuous metering device known in the art as a volumetric
proportioner and then deaerated. The resulting mixture was L,dn:,r~r,~:d to a carbo-
cooler where it was cooled and carbonated to approximately 2.5 volumes. The pH of
the finished beverage should be in the range of about 3.14 and the pH of the
prototypes was about 3.7. The finished product was then filled into standard 12 oz.
aluminum soda cans.
The nutritional profile and initial vitamin D3 Recoveries of the prototype low pH
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W O96/31130 PCTrUS96/04601
beverages in accordance with the invention are presented in Tables 23 and 24.
TABLE 23
NUTRITIONAL PROFILE OF PROTOTYPE BEVERAGE
SERVING SIZE 1 CAN (355 mL)
AMOUNT PER % Daily Value*
SERVING
Calories 0
Total Fat ~ 0 9 0%
Sodium 45 mg 2%
rOt~Ssil~m 25 mg 1%
Total Carbohydrate 0 9 0%
Protein 09 0%
Vitamin C 50% of RDI
Calcium 50% of RDI
Vitamin D 30% of RDI
* Not a siy"irica, IL source of other nutrients.
* Percent Daily Values are based on a 2,000 calorie diet.
TABLE 24
VITAMIN D3 (IU/KG OF PRODUCT)
(THEORETICAL FORTIFICATION A--810 IU/KG OF PRODUCT)
FLAVOR 0-TIME % RECOVERY
Cherry 597 73.7
Lemon Lime 613 75.7
Peach 701 86.6
Orange 580 71.6
Average = 76.9% vitamin D3 Recovery
CA 02217264 1997-10-02
W O96/31130 PCTrUS9'~ C01
EXAMPLE 4
CARBONATED BEVERAGE
An alternative embodiment of a liquid beverage concer,l,dle may be prepared
according to Example 1 or Example 2 excluding any i"g,~dienl~ other than the water,
calcium source, vitamin D3 and gum arabic (eg. the flavorant, and/or the cclDra,)l,
and/or the sweetener may be omitted). This liquid beverage concenl, dla may then be
combined with another liquid beverage concenl, dle, such as a commercial soda pop
concentrate, and the resultant blended beverage concentrate may ll,erearler be
combined with Cdl L ondled water, or combined with non-ca, L,onal~d water with the
resultant beverage being carbonated in the ",anner desc, iL,ed above in Example 3.
EXAMPLE 5
NON-CARBONATED BEVERAGE
A liquid beverage concenl,ale may be prepared by blending a liquid beverage
concentrate accor.li.,g to the prese,ll invention, such as described above in Exalllr'os
1 and 2, with non-carl,on-dled water. The resultant blend could then be placed into
aluminum soda cans, or light reducing bottles, the head space flushed with nil~uyen
gas or carbon dioxide to eli."i"dle oxygen which is harmful to vitamin and colorstability, and sealing the cans in the usual ",anner.
EXAMPLE 6
NON-CARBONATED BEVERAGE
An alternative embodiment of a liquid beverage concentrate may be pl~pdl~d
accon " ,9 to Example 1 or Example 2 excluding any i"yl eu;enl~ other than the water,
calcium source, vitamin D3 and gum arabic (eg. the flavorant, and/or ccloranl, and or
sweetener could be omitted), and thereafter t' s ndi.,g the concentrate with fruit juice,
vegetable juice, or any other suitable liquid matrix.
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EXAMPLE 7
POWDERED BEVERAGE CQNCENTRATE
The bill of materials for a powdered beverage concentrate in accordance with
the invention is p,~ser,led in Table 25.
TABLE 25
BILL OF MATERIALS FOR POWDERED BEVERAGE CONCENTRATE
INGREDIENT AMOUNT
Vitamin D3 Emulsion' 350 g
Calcium Glyceruphosphdle 291.6 g
Lactic Acid Powder (60% lactic acid) 181.3 9
Citric Acid 130.2 g
Natural Cherry Flavor 42.0 g
Sodium Citrate Dihydrate 19.3 g
Aspd, ldr"e 17.5 9
Ascorbic Acid 10.5 9
' This emulsion is des.i,ibed above with regards to batch 31.
A powdered beverage concer,l,~le was p,~:par~d by placing the calcium
glycerophosphdl~ sodium citrate citric acid lactic acid and ascorbic acid into the
chamber of an Aeromatic Top Agglomerator. The powder was then blended for two
minutes under medium fll ~ tion. The ter"perdlure was brought to 70~C, the
atomization was set at 1 Bar the alo",i~i"g nozle was placed at the highest level of
three p~s- !e positions and the fan capacity was set initially at 12 (nominal setting).
Aspa, lar"e was dissolved in approximately 800 ml of warm tap water and a
small amount of citric acid was added to achieve a pH of appro~i" ,al~ly 4. The vitamin
D3 emulsion and the flavor system were blended by hand with the aspartame solution
to yield approximately 1200 ml of liquid. The 1200 ml of liquid was placed on a stir
plate and agitated under medium agitation while being sprayed onto the fluidizedpowder for approximately three hours.
As the liquid was sprayed the powder became heavy and it became necessary
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to increase the fan capacity to maximum and place the alur,,i~i,,g nozle in the center
position. Per actual analysis a Kg of powdered beverage concenl~le conl~i"ed about
83.5 9 of calcium 12.9 9 of vitamin C and 31 900 IU of vitamin D3.
The final powder pa, liul~s were relatively large and brittle and were pulverized
before reconstituting with water. The powder was easily reconstitllted (see Example 8)
and flavor was typical of a powdered beverage conce, Ill ~le product without thecarbonation. Longer shelf life in this kind of beverage concenl,dl~ is ar,lic;~ d
because of the absence of water.
EXAMPLE 8
NON-CARBONATED BEVERAGE CONTAINING POWDERED BEVERAGE
CONCENTRATE
Approxi",alely 19.1 grams of the powdered beverage conce"l,dle
manufactured in Example 7 were ~ solv0d in a sufficient amount of tap water to yield
1 Kg of beverage. A Kg of the resultant beverage is pr~jected to contain about 1.4 9 of
calcium about 0.25 9 of vitamin C and about 607 IU of vitamin D3. As in the case of
the liquid form of the powler~d beverage concenl, d~e the acid system can vary
depending on the flavor s~l~.;te~l
EXAMPLE 9
POWDERED BEVERAGE ADDITIVE
A powdered beverage additive may be manufactured by the process desc, iL ed
in Example 7 containing at least vitamin D3 a calcium source and vitamin C but if
desired omitting sweetener acids flavoring etc. The resultant powdered beverage
additive could be added in applopriclle quanlilies to a liquid matrix such as a fruit juice
blend offruit juices vegetable juices coffee tea oranys~ le beverage. The
powdered beverage additive could be employed in bulk (eg. at an orange juice
processing facility) or on a serving by serving basis when provided in single serving
size packets.
It should be noted that if a liquid or powdered beverage concenl,~le or
beverage additive accordMg to the invention is intended for use in a liquid matrix that
may contain any dairy product (for example coffee or tea that may contain cream) a
salt of ascor i~ acid should be used in place of ascorbic acid to prevent curdling of the
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dairy product.
EXAMPLE 10
CALCIUM SUPPLEMENT
A calcium glycerophosphale/vitamin D3/vitamin C tablet suFplen,er,l was
pr~par~d by placing about 291.6 9 of calcium glycerophosphate and about 10.5 9 of
ascorbic acid (vitamin C) into the chamber of an Aeromatic laboratory batch
agglomerator. The powder was then blended for three minutes under medium
~git~tion. The temperature was brought to 55~C, the atomization was set to 1 bar, the
~lullli,illg nozle was placed at the highest of three po~~ ' !e posilions, and the fan
capacity was set initially at 9 (nol"i"al setting).
The perial~lLic pump was set at 7 cc/minute and app,uxi,,,~lely 350 9 of vitaminD3 emulsion was sprayed onto the fll ~ ed powder. The commercially manufactured
vitamin D3 emulsion desc, iL,ed above with respect to batch 31 was used in this calcium
supplement. However, any sl~'-''e dry b'enc'-~'e source of vitamin D, preferablyvitamin D3 or D2, may be used for making a solid calcium suFp'en,er,L accordi.,g to the
invention. As the liquid emulsion was sprayed, the powder became heavy and as
powderfluM;--~Lio" was depr~ssed the fan speed was incrementally i"cl~ased to 12over 55 minutes to ",ai.,Lai" medium flll; ~ ion. Te",peraL.Ire was also increased to
60~C after 16 minutes. After all the vitamin D3 emulsion was sprayed on the powder,
the heat was kept on and the powder was dried for three minutes. Per actual analysis,
a Kg of powder for tableting contained about 139.9 9 of calcium, 26.4 9 of vitamin C,
and 39,600 IU of vitamin D3.
The final powder particle was a soft agglomerate. No excipients were added to
the powder to f~cilit~te the tableting prucess. Using a tablet die of approximately 1/2
inch diameter, 600 9 of the final powder was col"pn:ssed using a Carver model C
laboratory press and an applied load of 200 pounds force. The tablet was easily
removed from the die. This process was repe~t~d using 1000 9 and 1500 9 of finalpowder to produce a total of three calcium supplement tablets, 600 9,1000 9, and1500 9, respectively.
A calcium suFplen,ent in solid form in accordance with the invention,
comprising calcium glyceruphosphate, vitamin D, and vitamin C, is believed to beadvantageous over prior art calcium supplements because it provides a source of
calcium that has a low aluminum content as well as providing vitamin D.
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APPENDIX A
A-1 VITAMIN D3 ASSAY
Refer to Figs. 1-7, eyd, I ,9 the pe, ru" "ance of this vitamin D3 assay.
I. OVERVIEW OF THE METHOD
The low pH beverage, the vitamin D3 emulsion and the powder beverage are
sapohi~ied with "lelllan-' - pot~c i~rn hydroxide to destroy the fat and releasethe vitamin D3 for exl, d~,lion. The Sdpol ,iried 5dl ", !e 5 are exl, dcled wlth an
elllel/penld"e mixture and the extracts are e\dpoldled to dryness using nitrogenand, ecor,-~lil,Jted with iso-octane. Sample extracts are eluted on a cleanup
HPLC column (cyanop,u~yl bonded silica), and column _~it~,h ,9 is used to
transfer a "slice" of the eluant cor ' ,' ,g vitamin D3 onto an ad.liliunal HPLCanalytical column (al " ,op,u~,yl bonded silica) for final qud"litdlion. The vitamin
D3 peak in the sample is qud"lildled using a linear rey,ession external ~Idnddldcurve.
Il. APPARATUS
A. General Apparatus
1. Centrifuge tube glass, 50 ml with teflon-lined screw cap
(Corex 8422A).
2. Centrifuge tube glass, 50 ml - conical (Kimax 45176).
3. Centrifuge (IEC Model Centra-HN or equivalent).
4. Water bath -capable of 40(i2)~C and 75(+2)~C.
5. Source of nitrogen (purity ~99.7%) - for evdpo,dlions.
6. Vortex mixer - S/P Mdyl leslil or equivalent.
7. Volumetric flasks - 100 ml, 500 ml.
8. Volumetric pipets - I ml, 2 ml, 3 ml, 5 ml, 7 ml, 15 ml, 30 ml.
9. Repedli"g pipet - "Tilt-a-Pet"
2-25 ml heads (VWR - 53481406) for ell,e,/l,er,ldi,e
2-1000 ml C, len" ,eyer flask reservoirs - size 24/40.
10. Repipet D;_pense, :, - Baxter-P4985 or equivalent
-1 ml for KCL solution (Baxter P4985-5)
- 5 ml for acelo";t, i!e (Baxter P4985-10)
- 6 ml for " ,ell ,anol (Baxter P4985-10).
11. Oxford Macro-Set Pipetter
(Baxter - P5079-2, or equiv; Qty =2)
1 - for sample transfer
1 - 4 ml for 45% KOH.
12. Therm-O-Vac - size 14/20 (Cole-Parmer #N-06140-15).
13. Teflon sleeves - sizes 24/40 (Cole-Parmer#N-06139-15).
14. Evapo-Rac E\ dpordlur for 30 mm tubes (Cole-Parmer
#N-01 61 0-35).
15. Centrifuge tube rack (Cole-Parmer #N-06737-40).
16. Cooling tray large enough to accG"""ocldle centrifuge tube rack
(#N 06737-40).
17. HPLC tubing - 0.040" sla;.,less steel - 2 feet.
~ 18. Caldnces - (a) MettlerAT200 (orequivalent) n ~d ' IEtoat
least 0.01 mg (for standards, vitamin D3
emulsion and powdered product.
(b) Mettler PM460 (or equivalent), ~ ~ ~' ~ ' 'e to at least
0.001 9 (for low pH beverage Sdll, 1~S).
19. Glass Stirring Rods.
20. Magnestir Stir Plate - Lab Line #1250 or equivalent.
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21. Teflon Coated Stir Bars - 2" length.
22. Beakers - 600 ml 800 ml 1000 ml.
23. C ~ r - Hewlett Packard-11 C or equivalent.
24. R~rl igerdlùr (freezer cor,lpdl l~ hl optional) for storage of :~Ldnddrd:~
at 4(+4)~C.
25. Lighting Peq~ ",el,b
Ultra-violet shields - F40T12 - Dayton Plastics Inc. for white
fluo,~scenL bulbs.
26. Scoop - 1/8 ~ pOOIl.
B. HPLC Instru, - ,enldliun
1. Columns: Guard (4.6 x 30 mm)
Cyano - Rainin - Cat. #CS-GU
Cartridge holder- Rainin - Cat. #140-200.
Cleanup - Chromegabond Cyano (4.6 x 250 mm 311 60 Ang~l,u,,,s) -
ES Industries.
Analytical - Hypersil APS ll (4.6 x 250 mm 3 1~, 120 Anstroms) -
Keystone.
2. Pumps: Two consld"l flow pumps capable of ope, dli"g at 5 ml/min
and up to 6000 psi (Beckman 110B with pulse dd""~ener
or equivalent).
3. Dt~ .lul:.: Cleanupsystem-fixedorvariable wa I l~hyll~
capable of ",onilc,ri"g 254 nm or 264 nm ( Waters
440 or equivalent).
Analytical system - Variable wa~/~lehylll detector capable
of ",on . i"g at 264 nm ~ 0.0025 AUFS. Under normal
ope,dli"g col, ~s the short term noise should be less
than 3% of the 5T vitamin D3 :.Ldnddl d peak height (Waters
486 or equivalent).
4. Injector: Alcott/Mi~;,ul"e,ili~ s 728 orequivalent.
5. Column Oven: Capable of 35~C - 100~C and + 1.0~C settings andaccuracy. Storage for 2 x 250 mm columns and one
30 mm guard column.
6. SY.jtCI ,9 Valve: HPLC column s~. it~;l, ,9 valve with at least 6
ports. Has a working range up to 6000 psi
(Mi~ilu,,,eriLi~;~ 732 or equivalent).
7. Recorder: One 10 mV ~bcordi"g device for the cleanup HPLC output
and either a ,~co,der or an illL~:yldLul for the analytical
HPLC system. A data system capable of l"oniLori"g
acquiring and ,~p,ucessi"g two chal-nei~ of data is
strongly ,~cor"",ended.
8. Solvent Reservoir: 10 Liter - Common to both cleanup and
analytical HPLC systems (VWR #KT953901-
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W O96/31130 PCTrUS9G/~SC01
1003 or equivalent)
Ill. REAGENTS
A. Standard Rer~l~nce Material - Vitamin D3).
1. USP ,~rt~ nce sld"dd,d #1310 (Ch~ rul = vitamin D3).
Consult current USP literature for the current lot number. Potency =
40 000 IU per mg. Store at 2~C to 8~C. Care must be used in
opening the sealed ampules to avoid introducing glass r,dy",~"L~ into
the !~ldnddl d. Vitamin D3 must be used from an open ampule
i"""ed;at~ ly and di~.cd,ded.
B. Chemic31s
1. Amyl Alcohol Analytical Reagent r~co"""end M " Icklud
UN 1987.
2. 1\1e;1ldl1ol HPLC Grade ~ cor"",end Burdick & Jackson #230.
3. Iso-octaneHPLC Grade ~ecor"",and Burdick & Jackson
#362.
4. PentaneHPLC Grade ,t:co",lllend Burdick & Jackson #312.
5. Diethyl Ether Anhydrous ,~co"""end M " luhludL UN 1155
6. Potassium Hydroxide 45% solution ~ecor"",end Baker#3143-03.
7. Sodium Ascorl,dL~ RecGr"" ,end Aldrich #26 855-0.
8. Acelor,- ;'~ HPLC Grade ,eco"""end Burdick & Jackson
#015.
9. Ch' ~ ~Orul,,, HPLC Grade , ~co" " "end Burdick & Jackson
#048
10. Potassium Chloride Reco"""end Mal'- lulhudl#6838-500~NY.
11. n-Butyl Chloride HPLC Grade rt:cGIllll~and Burdick & Jackson
#034.
C. Solutions
1. HPLC Mobile Phase
Vol~""~L, - -'Iy pipet 40(_0.1) ml of n-butyl chloride 20(_0.1) ml of
amyl alcohol and 10(_0.1 ) ml of ch' o~. ", into 4000 ml of iso-octane.
Mix well. Make four liters at a time - roughly equivalent to 1.0% n-
butyl chloride + 0.5% amyl alcohol + 0.25% ch'~rurur,,, in iso-octane.
Use for both cleanup and analytical HPLC systems. Cor" 'e: ~y fill
the 10 liter reservoir prior to each day's analysis.
2. Exll d~ liun Solutions
#1 - 20:80 ell,er/~nldi-e: Mix 200 ml of diethyl ether with 800 ml of
pentane. This is sufficient for up to 20 Sdll, es (2 X 25 ml per sample
is required). Prepare fresh daily.
#2 - 33:67 t:ll ,erl,l enldne: Mix 250 ml of diethyl ether with 500 ml of
pentane. This is sufficient for 28 Sdll, !e s (25 ml per sample is
required). Prepare fresh daily.
3. KCL Solution
Prepare a 10% KCL solution using distilled water. Mix 50 9 of
pOpc~illrn chloride with distilled water and dilute to a 500 ml liter
volume. Store at room ~el,,perdL.Ire and e. dlion date is one month
from date pr~pd,~d if kept tightly capped.
D. Pl~pdldLion of Vitamin D3 Stdl~ddl~ls
NOTE: Work under UV-shielded, whife, fluorescent bulbs wifh the ulfra-
violef shields described in Secfion 11.25 if possible. If unprotected white
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W O96131130 PCTrUS~6/01C01
lights are used, extra precautions must be taken to keep an solutions of
Vitamin D3 protected from light by covering containers with aluminum foil or
by using amber low-actinic yld s~ .~ c.re. Sl~ndah~:, are also heat sensitive
and should only be briefly removed from the refrigerator for immediate use.
NOTE: Due to the ",ell,o.l'~- suscel ' ' ' 'y to low level conldn ,a~ , an
volumetric flasks must be rinsed with iso-octane prior to preparation of
Vitamin D3 standards.
1. Stock Standard (AppluAilll.~t~ly 1,920 lU/ml)
Weigh 24(+1) mg of vitamin D3 into a 500 ml volumetric flask.
Dissolve and bring to volume using iso-octane. F-, - dlion date is two
weeks and it must always be stored at 2~C to 8~C when not being
used to prepare the i"lt:r",edidl~ :~ldl1ddl-1.
2. I"L~""edi~le Standard - ISTD (AppruAi,,, ~t~ ly 27 lU/ml)
Pipet 7.0 ml of stock :~ldnddld into a 500 ml volumetric flask. Dilute to
500 ml with iso-octane. ': ~: dlion date is 10 hours for plepdldliùn of
the working :~ldnddld~, but can be used for 2 months (at room
l~l I "~erdlure) for 1~ - h ,9 the, el~l ,lion times on the cleanup
HPLC system.
3. WorkingSI~nddld~-3T 5T 15T 30T
lion is 1 week. Store at 2~C - 8~C).
3T = Pipet 3.0 ml of ISTD into a 100 ml volumetric flask and dilute to
volume with iso-octane (d~J~JIuAilll~ly 0.8 lU/ml).
5T - Pipet 5.0 ml of ISTD into a 100 ml volumetric flask and dilute to
volume with iso-octane (app,uAilll..~ly 1.3 lU/ml).
15T = Pipet 15.0 ml of ISTD into a 100 ml volumetric flask and dilute
to volume with iso-octane (app~uAill..~t~ly 4.0 IU/ml).
30T = Pipet 30.0 ml of ISTD into a 100 ml volumetric flask and dilute
to volume with iso-octane (a~u~JluAilll._~ly 8.0 lU/ml).
IV. PROCEDURE
Sample Pl erjd, dlion
1.a. Low pH Beverage (300 IU/KG - 900 IU/KG)
Accurately weigh (to the nearest 0.001 g) 12.5 9 of the low pH
beverage into a 50 ml centrifuge tube (Corex #8422A) and
proceed to #2 in the Saponir,. dliun Section.
b. Vitamin D3 Emulsion (-100 00 IU/KG)
Accurately weigh (to the nearest 0.0001 9) 0.19 of the bulk emulsion
into a 50 ml centrifuge tube (Corex #8422A). Add 10 ml of
d i~ - I/de.or,i,e water. Proceed to #2 in the Saponirudlion Section.
c. Powder Product (-35 000 IU/KG)
Accurately weight (to the nearest 0.00019) 0.39 of the powder product
into a 50 ml centrifuge tube (Corex #8422A). Add 10 ml of
distilled/deiol ,i~e water. Proceed to #2 in the Saponircalion Section.
2. Add about 0.4(~0.1) 9 of sodium ascorl,dL~ (1/8 level teaspoon)
and vortex 10 seconds.
NOTE: Sfart with a low vortex speed and increase vortexing speed
with each sLIcces.civc step (i.e., affer the addition of methanol, and
again affer the addition of 45% KOH).
3. Add 6(+0.3) ml of methanol and i" ""edicl~ly vortex for 15 seconds.
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4. Add 4(iO.3) ml of 45% potassium hydroxide solution tightly cap
the tube, and i"""edi~t~ly vortex for 20 seconds.
5. Place the tubes in a p,t:hedL~:d water bath at 75(i2)~C for 30
minutes. The tubes should be vortexed for 5 seconds at the 10
and 20 minute intervals.
6. After 30 minutes remove the tubes from the water bath and
place in ice water for a minimum of 30 minutes to bring them
rapidly to room l~r"~.e,dLure.
EAII d~il~n
7. Add 5(iO.3) ml of ac~:Lunil,.' to each tube, cap and vortex at a
" ,oderdlt: speed for 5 seconds.
8. Add 25(i1) ml of 20% ether/80% pentane mixture and shake in
a wide ser, ' ' cular arc across the front of the body 20 times.
Invert the tubes with each stroke.
9. Briefly centrifuge at ",ode~dL~: speed (app,uAi",~ ly 300 x G for
1 minute) to COI" ' : layer sepd,dLion.
10. Draw off the clear ether/,~,enldi)e using the si~hor, ,9 apparatus
and vacuum. (See Figure 1). Transfer the top layer to a 50 ml
conical centrifuge tube (Kimax #45176) leaving behind 4 to 8
",'"' "ele,:, of the ell,er/,~,enldne mixture. Avoid transfer of any
middle layer or aqueous (bottom) layer.
NOTE: If any of the middle or aqueoLIs bottom /ayer is accidentally
l,dn~-r~"~d, the sample must be discarded and the assay r~,pe~tf~d
11. To avoid sample to sample cGr,ld",' ,alion rinse the si~,hon' ,9
apparatus with 5 - 7 ml of pentane and add this rinse to the
dm,~
12. Evaporate the L~dnsrt~ d ~II,er/per,ldne layer in the warm wate
bath (40 i4~C) with nitrogen to about 2 ml to allow for e~ nal
l,dnsre, ,. (See Figure 2 for the Evapo-Rac e~,dpordlion
apparatus.)
13. Repeat the ~Xtl d~liol- in steps #8 - #12 once cor, ' :. ,' ,9 the
extracts in the same 50 ml conical centrifuge tube.
NOTE: Be careful not to overflow ~he 50 ml Corex centrifuge tubes
with the 2~ ml eAl,dclion solutions (#1 or#2) during the 2nd and 3rd
~l,d~,lions. Should this occur, the sample mustbe discarded and the
assay repeated.
14. For the third eAIId~;lion, follow steps #8 - #12 using 25 ml of 33%
ether/67% pentane solution (not the 20% ether/80% pentane solution)
15. Evaporate the co",' ..,ed ~AIId.;lions to dryness. Remove the
centrifuge tubes from the water bath as soon as e\,dpordlion is
col " !~: The extract should appear clear or as a white or slightly
yellow film. Make sure that the extract is cor" ' ' 'y dried before
~ ~con:,lilution. The tubes may have to be gently tapped to CGI I l, ' ~ '
the e~/apordlion.
16. I~ edidhly r~con:,lilute with 2.0 (iO.006) ml of iso-octane with a
class A volumetric pipet. Be careful to thoroughly rinse down the
walls of the tube. The tube should be tightly capped to prevent
e\,dpoldLion and vortexed 5 seconds to mix.
17. Finally, add 1 ml of the KCL solution to each sample and touch to the
vortexer briefly to mix. Tightly cap and centrifuge at moderate speed
(app,uAi",c~ly 300 X G) for 1 minute to con, '~t phase sepa,dlion. If
using a centrifuge equipped with a s~ ;"g; ,g bucket rotor place the
centrifuge tubes on the outside perimeter of the rotor. This is to
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WO96/31130 PCTÇUS9''01C01
prevent the conical tubes from brt:al~i"g. Transfer only the top layer
to a vial and tightly cap. Be careful ~ to transfer any of the
saturated KCL solution.
NOTE: The sample exfract must be analyzed within 24 hours from
fime of p,t:pa,dlion. If fhe HPLC system encounters problems, the
aufc san, '~r vial should be immediately stored below 8~C affer
pr~:pardlion for up to 48 hours. No sample can be reinjected affer an
aborted HPLC analysis if it was leff in the auLusai"N~r at room
temperature ovemight.
18. inject onto the eq~ b.dlt:d HPLC system (section V).
V. HPLC CONDITIONS
A. Cleanup HPLC System - See Figure 3 for configuration.
1. Column: Ch,u,,,egabond Cyano (4.6x250 mm 31~) with CS-GU
guard column (4.6X30 mm).
2. Eluant: 1.0%1-chlorobutane + 0.5% amyl alcohol + 0.25%
cl ,'c uror' ' H n iso-octane.
3. Run Time: Slice dele", laLion =app~u~illl~tuly20 minutes.
4. Flow Rate: 1.5 ml/min.
5. Injection Volume: 25û~1.
6. Column Heater: 40(_1)~C.
7. Detector: 254 nm or 264 nm.
8. Recorder~ y, dLùr or data system (pl t:rel l ~d).
9. Column Actuated by timed control from injection point to
Switch: c - n. Slice time window should be no greater than
1.0 minute (with 0.1 minute accuracy) for c~ n of vitamin
D3.
B. Analytical HPLC System - Figure 3 for configuration.
1. Column: Hypersil APS ll (4.6X250 mm, 3~).
2. Eluant: 1.0%1-chlorobutane + 0.5% amyl alcohol + 0.25%
ch'~ uru"" in iso-octane.
3. Run Time: Approxi",at~ly 35 minutes.
4. Flow Rate: 1.5 mVmin.
5. Column 40(_1)~C.
Heater:
6. D~L~uLion: 264 nm ~0.0025 AUFS, (Waters 486).
7. Recorde!: Reco"""end the use of an i"Lt:y,dLur or data system for
reprocessing.
8. Frll ~ b.dLt: the columns and obtain a stable te s ~ ,e. Inject the
i"L~---,edidLt: ~Ld"cla,d ISTD (no column switch) at least 3 times
until a consi:,L~r,L r~tenLion time (r~:LenLion time +0.02 minutes) is
e~ - -ed on the cleanup HPLC (Figures 4 & 5). Always verify
the cleanup HPLC ,~ :L~r,Lion time within 1/2 hour before analysis
of :,Ldnddl ds or Sdl I, ' C The run time is appruxi" ,~ly 2û
minutes however the time required to eq~ b.dL~ the columns
with fresh eluant is dppl u~ tuly 2 hours.
9. After d~Lel " , ,g the ~~l~"Lion time of the il lL~I " ,edidl~ Ldn-.ldl d ISTD
on the cleanup column set the slice window (i.e. transfer of vitamin
D3 from the cleanup column to the analytical column). This is done by
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W O96/31130 PCTrUS96/O~G01
setting the s~r ;~ h ,9 valve to switch the vitamin D3 from the cleanup
column to the analytical column at 0.10 minutes before the vitamin D3
first elutes from the cleanup column until 0.10 minutes after the
vitamin D3 peak returns to '~ c s -' Ie on the cleanup column. See
Figures 4 and 5. Slice window times should not exceed 1.0 minute -
using a minimum among of time (genel 'Iy 0.8 - 1.0 min.) necessaly
to collect all the vitamin D3 while preventing the transfer of any other
illl~ re~ g cGnll)onent~.
See Figures 6 & 7 for the cleanup and analytical HPLC
ClllUllldlUyldlllS of a 15T working :~Ldl~ddll~.
Vl . HPLC ANALYSIS
A. Upon verifying eqll ' b.dLion of the HPLC system and e -~ 1 '' lg the
- " n window, inject three (or four) working :~Ldnddlds (3T, 5T,
15T, 30T) and then the sample extracts. The three (or four) working
sLdl Idards should be injected once again at the end of the run.
Only single il l, - ns of each sample are required.
Vll. CALCULATIONS (Use only peak heights for reporting purposes)
Note: Peak height is required for quantitafion as small amounts of ~ass ' ,e
noise can cause large area differences.
A. C ' llation of Working Standard Concel lL~ dLions
1. C-'~"'-l~ the concel lLl dLion of the vitamin D3 in working :,Ldn.ldl d~
3T, 5T, 15T, 30T from the F~ ;. lg equation:
lU/ml = (W) (P) (7) (PV) = (W) (PV) (0.0112)
(500) (500) (100)
where: W = weight of vitamin D3 ~Ldnddl .1 in mg.
P = 40,000 lU/mg for vitamin D3
PV= final pipet volume for working ~Ldnddld~. ~
= 3for3T
5 for 5T.
= 15for15T.
30 for 30T.
Example: for a 5T sldnddl d p, t:pdlt:d from a stock solution that
con ~ Ied 24.00 mg vitamin D3, the concerlLIdLion is c?lclll-~-d as
follows:
lU/ml = (24.0ûi r4Q.Q00) (7) (5) = 1.3440 lU/ml
(500) (500) (1QQ)
- B. Ca' ll~tion of the Standard Curve Using Linear Reyl~ssion and the
Qudl,LiLdLion of Vitamin D~in Samples
1. The peak heights of each It:spe-.Li~/e level of the working :,Ldnddl d are
averaged. A lineam~yl~:ssion line is c ~cll~-~od by using the average
peak heights (y-axis) and the concenLIdlion (x-axis) forthe It:s,cecLi~e
working :~ldllddr~J.
Example: A lineam~:ylt:ssion line (vitamin D3 peak heights versus
concel,L,dLiol-) for264 nm channel is pl~serlLed below. Two ill,- -ns
(b~g l n lg and end of run) were made per each level of working
:,Ldndal d.
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Working ~ ~onc. ~ lo. Avg.,Peak
IWl~nl i l~ ~ject Heigh t
5T 1.3440 2 2.6396
15T 4.0320 2 8.0789
30T 8.0640 2 16.5839
Slope = 2.07775
y-i, lk~ JL = -0.20754
Corr. Coef. = 0.99994
2. The 5dlll les should be qua"LildLed by L,ldul~ 9 the :-ld"darda
around the 5dl l l 1 ?~
C. Low cH:BeveragQ.and Vitamin D3 Emulsion. and Powder Product
C~ tinn
1. Per Weight Basis - IU/Kg:
Vitamin D3 (lU/kg) = ~C) (V) (1000)#
'(S) (X)
where: C = Vitamin concellLIdLion (lU/ml) from sLdllddld
curve.
V = Volume (ml) of iso-octane to l~con:,LiLute
extracts.
1000 = converts grams to k- -_ dlll~
S = Sample size in grams.
X = 0.86 for 75~C sa~.or,iricdLion factor for
thermal isomel i~dLion of vitamin D3 to
previtamin D3.
# = Sl Ihstitl ltp 100 for 1000 to convert to lU/100g.
Example: A 12.533 9 (S) low pH beverage sample was ~e- on:,LiLuted in
2 ml (V) of iso-octane which gene,dL~d a peak height of
6.2330. The co"~:,por,di"g vitamin D3 concer,L,dLiol1 (C)
cL ~ Ied from the previously c~cl~ -d ~Ldncldl.J curve was
3.0998 lU/ml. The final concerlLIdLioll would be ~ t-d in
the / i.lg manner:
Vitamin D3 (lU/kg) = (3.0998) (2) (1000) = 575 lU/kg
(12-533) (0.86)
A-2 CALCIUM ASSAY
The Simultaneous Determination of Calcium (Ca) in a Low
pH Beverage by ICP-AES Using a High Solids Nebulizer
A. THEORY
1. Inductively coupled plasma atomic e~ sion spe- LIu,ll~L,y (ICP-AES)
is an atomic speuLluscopic technique that has several advd"Ldges
CGI l lpdl ~d to atomic aL,sol ~,Lion: P'~ ,L cleL~uLion limits a broad
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linear dlion range of over four orders of magnitude for most
elements, minimal il,telr~:r~nces and the ability to del~rll, ,e several
ele. "er,L~ in the sample simultaneously under one set of operdLi"g
co~ s. These advdllLdges lldllsldLt: into less sample pl~pdldlion,
~ I b. dtion and analysis time for the analyst.
2. The ICP-AES instrument consists of three cG"")onellL~. sample
introduction device, Iul- hbux and spe- I,ur"eL~r. Most cor"")only
Sdl l l !es are introduced in the form of solutions which are neh~ ~d
(broken into tiny droplets) and passed into the torch with a stream of
argon. In the lur.:l)bux 1-2 kW of radio-frequency power is coupled
from a copper coil (inducfor) into a small region inside a quarlz tube
(torch), through which argon flows. The power density in this region
is high enough to heat the argon until it ionizes and, since the region
is at dll "osphe, ic pressure, there are sufficient cc ~ens with other
argon atoms to instantly ignite a plasma with a temperature of about
10,000 K.
3. The ".i- ur,l~L~l-sized droplets from the nebulizer enter the bottom of
the torch and pass through the cooler (6000 K), darker central region
of the plasma called the axial channel. Here water is evd~JordLed, and
the l~rr , ,9 dry pdlLicles of analyte are vdpo,i~ed and dLu",i~ed
(",-'e ~ ~les broken down into atoms) by the heat of the plasma in just
a few 1" o - e nds. FYcit-~'ion and ioni~dLiol~ of the outer ele~ LIuus of
the atoms occurs; the intensity of the e r";~s;on that results from the
dePYici~ on of these atoms and ions is p, upu, lional to the
conce"l, dlion of analyte in the original solution. Thus, ~ b. dlion
consists of measuring the intensity of analyte er"-s n for slandd,ds
of known concenL,dlion.
4. Light emitted by the ICP is ~ C'lf ~ d by a lens in the s~.e..l,u",eLer and
focused onto a dirr,d-.lion grating which di~pe,~es the light into its
cor"l,onenL wav~l~nyll ,:,. The emitted I ddidliun u a~elenyl h resolved
from all the analyte ele."e"l:. is c-- sr: d sim~ neously by several
del~ulul ~ placed in front of the grating and converted into an ele~ Irical
signal. A data system relates these signals to the concenl,dtions of
the ~le."enl:, in the sldnddld:~ and ~ 5 the analyte
conce"l,dlion in the Sdll, ' S
5. The particular instrument used in this method features a movable
ehl,dnce slit conl,.- ed by a high ,~:s ~ tion stepper motor called
SAMI (Scanning Accessory for Multielement Instru",er,ldlion).
Moving the entrance slit slightly changes the angle of inc;dence upon
the grating, and slightly changes the wavulel,yllls incident upon the
exit slits. This feature allows the user to perform bachyluund
co"~ulion in the sample matrix by sLIL,lldulill9 the e",;~sion
bachyl ound just off the peak center.
6. This method employs a speedy dilution pr~pd, dlion of Sdl l e S with a
~ surfactant and dilute acid. A special kind of nebulizer, called a
maximum diasolvcd solids n-h~ : (MDSN) or high-solids nebulizer
is required to provide long term operdlion without clogy; ,9. Rec~use
the viscosity of :,ldnddl.ls and Sdll, les iS quite dirr~ r~"t an internal
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aLdnddld must be used to co",~ensdle for the poorer neb~ tion
erri..;en-_y of the high solids Sdl ", !e ~ Cobalt is added to each
sLdnda,d so that they are exactly 20.0 mg/L Co. C; ' b.dLion consists
of measuring the analyte/Co ratio in the standards as a function of
analyte concenLIdLion. An exact quantity of cobalt is added to each
sample so that if they were diluted to 50.0 mL, their cobalt
conce"L,dLion would also be 20.0 mg/L. Note, however, that the
analyte/intemal sLdndd, ~J ratio in the Sdl,, 'e s will not change with the
total volume, and so volumetric ware is not necessaly for the sample
pl epal dLion. When the SOr - a. e asks for the "sample volume" to
A a dilution factor, the analyst should enter 50 mL, the volume
that would make the conce,lLIdLioll of cobalt in the Sdl~, ' s equal to
that in the aLdnddlda.
B. MATERIALS
1. Instrument
a. Inductively Coupled Argon Plasma C., I;;,aiOn Spe-_L,u"leLer, ARL
Model 3560 or Accuris
b. Ryton V-groove n~bu . ARL#173259-0000 or Precision
Glass #510-50 only
c. Spray chd"lLe,. ARL#173142-0003 or Precision Glass #110-34
or equivalent
d. ICP torch: ARL#1390û9-0003 or Precision Glass #100-05 or
equivalent
2. General Labu, dLùly Ftll 'i, l~enLlFc fi ' ' ~ s
a. Analytical balance
b. Fume hood
c. Di "c ~ ~'e, flat-buLLur,,ed, 50 mL plastic centrifuge tubes with
caps (Baxter C3902-14 or equivalent)
d. Plastic coated rack suitable for holding many centrifuge tubes
e. 125 mL, 250 mL, and 1 L plastic bottles for storing sLdndd,da.
polymethyl~.~, llene (PMP) or equivalent
f. D;spc--'-'- plastic transfer pipets-3.5 mL capacity
9. E ppendo, r pipet or equivalent, 1000 I~L capacity with tips
h. 50 mL 1~ r:. ~t~rorequivalent
i. Plastic di_penser bottle (PMP or equivalent) fitted with a Teflon-
constructed di_penser top with arlju-' ' ' volume between 1-
10mL; dispenser may be fitted to concer,L,dLed HCI bottle
directly
j. Magnetic stir plate and Teflon coated l"ay"etic stir bars
k. 1 L and 250 ml volumetric flasks: glass or plastic (PMP or
equivalent)
I. Class A volumetric flasks: 2,4,5,10,15,20,25,40,50 mL
m. Options: 1 mL digital pipet with tips, Rainin EDP-Plus or 1 mL,
glass volumetric pipet or equivalent
3. Chemicals/SLdndd,-ls
Unless otherwise noted, the r 'lO.~ g chemicals should be stored at room
temperature. Their expiration date is one year affer the date they are frst
opened. Upon ~Yp.. diiun the chemicals must be either discarded or re-
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evaluated.
a. High purity stock sLdrlddld solutions (NIST or NlST-L,~ e)
10 000 mg/L Ca 10 000 mg/L Co 1000 mg/L Co. These stock
aLdnddl d solutions expire on the date given by the manufacturer.
b. Hyd,u..l1 ~ric acid J.T. Baker BlA-grade or equivalent
c. Triton X-100 Kodak scintillation-grade or equivalent
d. Argon gas minimum 99.996% purity
e. High purity water ~ 'i, - ~-treated or equivalent
c. iNSTRUMENT~t OPERATIN~ ~ON~ITl~)NS
1. The -~ lenyll,s that have been used are listed in the table below.
The instrument should be installed with identical chdnnt l. if p~ s ~ !~
be~o~ ~se the sensitivity of the line and the pc s ~y of i"l~:~ re(ences
can change if a different line is e", '~,/cd for analysis.
ELEMENT WAVELENGTH (nm) TYPE ORDER
Ca 317.93 ion 2
2. Typical ranges of operdLi"g conclilions for the ARL 3560 are listed
below.
a. Incident power: 1200-1400 watts
b. ~fl~ct~d power: ~5 watts
c. Snout argon gas flow: on
d. Coolantargon pressure: 30-40 psi
e. Plasmaargon pressure: 20-30 psi
f. Nebulizer argon pressure: 30-46 psi
(-0.6-0.7 Uminute if a mass flow conl,. -. r or other type of
flu~nlelt:r is used to regulate flow)
9. Pe,: pump flow rate: dial setting which cor,~aponds to
-2.5 mUmin. (dependa on make and model of pump)
h. Fe~ i ~ pump tubing: 1.12 mm l.D. red/red P.V.C.
1\1_.ul~ne orequivalent
3. Software pdl dl I I~L~:ra. These are stored in the TASK files which
perform e ~.dLion and sample measurement and must not be
altered.
a) InLeyldlions (on-peak): three 5 second i"l~:y,dLions per sample
b) InLt:gldLions (off-peak): two 5 second illLeyldLions taken at-80
SAMI units off peak center
D. STANDARD AND SOLUTION PREPARATION
Store at room temperature unless ull,en~;se noted.
1. 2% HCI rinse: Mix conce"L,dL~d HCI with high purity water in the
appruAi",dle ratio of 20 mL acid to 1000 mL total volume of solution.
Use a plastic COI, ,er of a size app,up,idL~ to the volume of solution
p,~pdlt:d. For exd", !e to prepare 20 L of 2% HCI fill a 21 L carboy
with high purity water to the 20 L mark and add 400 mL HCI to the
water. F-, ..dLion: 6 months.
2. Triton X-100 solution (appru,~i" Id~ly 5%): Add about 700 mL high
purity water to a 1 L plastic bottle con ,i"g a Teflon-coated stirring
bar. Place the bottle on a ",ay"etic stirrer and begin stirring at a
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Illode~dl~ speed. Slowly add 50 mL Triton X-100 from a graduated
cylinder. vVhen the Triton is .li;.:.olved, fill the boKle dppl U~il ,..Jtuly
1000 mL with high purity water. Transfer to 1 L plastic boKle fiKed with
a Teflon-constructed .li~panser with - 5 ~-'-' ' -. volume from 1-10 mL.
F- dLi~n: 6 months.
3. Tld.litional :~Ldnddld solution pl~pdldliun Prepare 1 literofthe
d,u~luplidl~ sldnddld solution. Add the i" "- ' ' amount of 10,000 or
1000 mg/L stock :~Ldllddld solution to a 1 L volumetric flask using a
Class A pipet. Then add app,u,~i",~.~ly 800 mL of high purity water,
2.00 mL of 10,000 mg/L cobalt internal :,Ldl Iddl d (Class A pipet), and
20 mL (r~ F ~ or di~",enser) of hyd, u~ loric acid to each flask.
Add 50 mL of Triton X-100 solution from the di "~enser to each flask
and then fill the flasks to volume with high purity water, slowly to avoid
forming suds. Agitate well and transfer to clean, dry 1 liter storage
boKles. Dispense as needed into 125 mL storage boKles to use at
the instrument. F-, dlion: 6 months.
4. Standard blank solution: Prepare 1 liter of a sldndard blank at the
same time, and from the same reagent batches, as the above
~ldnddl d:~. Add 800 mL high purity water to a 1 L volumetric flask;
2.00 mL of 10,000 mg/L cobalt internal sldnddld (Class A pipet), and
20 mL (~F, ~$ 3r) of hy.l,uch'-ri., acid to the flask. Add 50 mL of
Triton X-100 solution from the di~."enser to the flask and then fill the
flask to volume with high purity water, slowly to avoid forming suds.
Agitate well and transfer to a clean, dry 1 liter storage boKle.
Dispense as needed into 125 mL storage boKles to use at the
instrument. ':., dliOI) 6 months.
5. Intemal ~Ldllddld ~t:r~,~nce blank solution (ISRB): Prepare 1 liter of
an ISRB at the same time, and from the same reagent batches, as
the above "ldnddl d~. Add 800 mL high purity water to a 1 L
volumetric flask and 20 mL (l ~ F i~ ctt~r) of hy.l, u.,L' - ric acid to the
flask. Add 50 mL of Triton X-100 solution from the di~penser to the
flask and then fill the flask to volume with high purity water, slowly to
avoid forming suds. Agitate well and transfer to a clean, dry 1 liter
storage boKle. Dispense as needed into 125 mL storage boKles to
use at the instrument. F- ~ dLiOn 6 months. Note that the ISRB
does not contain cobalt, but the :~Ldl Iddld blank does. The ISRB is
analyzed before any :~Ldl Iddl ~ or S'dl I, !e i, the purpose is to subtract
the intensity of analytes found in the ,~agenl:, (Triton X-100 solution,
HCI, and water) from the analyte i"l~nsilies found in the sldllddld:~
and Sdll,
E. PROCEDURE
1. Standard I la, l'" lg
a. All boKles used for storage of standard solutions must first be
soaked in 10% (v/v) HCI for a minimum of three hours, followed
by multiple rinses with high purity water. Air dry or rinse several
times with the :~Ldl)ddld. When reusing the boKles for a new
batch of the same :~Ldl Iddl d, no acid soak is necessary - simply
rinse several times with high purity water and then several times
with small portions of the fresh :~ldnddld.
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b. As the working ~Idnddrd:~ in the 125 mL bottles are used up,
simply refill the bottles from the 1 L ~Idnddlds prt:pared in D. 3.
c. Because there are many Sdl ", !e ~, the most efficient way to add
the cobalt internal sldllddld to the Sdlll, 'es is with a 1 mL digital
pipet.
2. Sample Pl ~Jdl dlion
a. Refill the reagent containers before p~:pdli"g Sdl~ ~ '-s so that
the same batch of r~agenl~ can be used for all Sdlll,''_S and the
blank.
b. Remove the caps and arrange the empty 50 mL tubes, with
labels, in the rack b -g- "~;"9 with the sample blank and two
tubes for each sample.
c. Transfer sample to a plastic storage container. Place these
cor,' ~ ,el:, directly on a Illayll~lic stirring plate and add a Teflon
coated stirring bar. Set the stirrer at an i, llel 1 l ,edidlt: speed.
After a minimum of one minute of ayildlion begin to withdraw the
sample for v. ~ I, ,9 with a ~ - - ~ '~ 'e plastic transfer pipet.
d. Carefully weigh and record to the nearest 0.0001 9, 5 g of
sample into the plastic tubes. The sample blank tube is left
empty at this point. Add the ~ ;"g r~agenl~ to each tube,
including the blank, in this exact order:
(1) Add 2.5 mL of Triton X-100 solution using the d;_penser
bottle.
(2) Add app,~.Ai", ~ly 45 mL of high purity water
(3) Add 1.00 mL of the 1000 mg/L cobalt internal :,ldn-ldl .I with
either a - - ' dl~d digital pipet (p,t:r~r,e:d) or a Class A
pipet.
(4) Add 1 mL of conceril,dLed hyd,uch' ic acid with an
Cppendo, r pipet or from a Teflon di~penser bottle.
(5) Add high purity water until the total volume in each tube is
app, uAill,at~ly 50 mL. Put the caps on and shake the
tubes thoroughly.
3. Instrumental Analysis
a. The f. " . ;. ,9 instructions refer to some general cha, d~;Leri:.Lics
of the PLASMAVISION SOri dlt:, which is currently used on all
instruments running this method, but no attempt has made to
describe specific key sequences needed to perform these
procedures, since that i,,ru,,,,dLion is provided in training.
Equivalent ope, dLions must be pe, rur" ,ed with other versions of
the SOr~
b. Turn on the plasma and allow a thirty minute warm-up time
before r-''b dlion. Turn on the computer and printer and start
the SOri ~ . Begin pumping 2% HCI rinse solution through the
nebulizer.
c. Perform an instrument configuration before the first caGrdliun is
made for each 8-hour shift. This will check computer-instrument
commu"i.,dlions and check the SAMI motor. Watch the motor to
be sure that it turns properly.
~ d. Check the optical ' _ " "enl using the 150 mg/L calcium
:~ldnddl d. This procedure will insure that the SAMI motor is
operating properly and that the - ' b.dlion will always be
pe, ru, l l ,ed near the exact center of each analyte peak. Perform
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this procedure before the first --' b.dLion is made once during
each 8-hour shift. Choose appl ou, idLe setup options and then
run the profile. The measured peak centers for the element to
be measured must be within +6 SAMI units of the current SAMI
profile position. If this result is not oL: ~ ,ed consult the
supervisor: either a new default SAMI profile position needs to
defined, or the instrument requires service.
e. Select the apprupridle task and the applupridle calibration
sequence file name and begin '-b. ~Lion. Aspirate the :~Ldnddl d
solutions into the plasma starting with the ISRB solution
plepd,~:d in D.5. The software prompts for each sldnddld by
name. Be alert to any error l"essages If an error occurs, write
down the ",essage and consult the supervisor. After the last
sLdl)ddl d has been run save the data and have the soft--- .b
C~ h te a linear ,eyl ession for each element. Print the
r-' ~.dLion data, which su"",ldli~es the element illlensities in
each sLdllddld the colleldLion coerri~ "L, and the c-'c~ d
conce"L,dLions of the ele",eril:- in each :,ldnda,d.
f. Enter the section of the Sur~l a~ e to analyze the Sdl " le s Set
the print options to print whatever docL""elr,ldlion is required.
Select a name for the file that will store sample results; if no file
name is chosen, the proy,d", will store the data under the task
file name by default.
9. Shake the Sdl " ~ 'e s i" " "ed;~ly before introduction into the ICP.
h. HIT THE ENTER OR RETURN KEY AFTER ENTERING THE
WEIGHT, VOLUME AND NAME OF THE SAMPLE. Note that
the PLASMAVISION software prompts for the sample weight
and volume before the sample is introduced and the sample
name after it has been analyzed. The dilution volume for
Sdl, !e s will always be entered as 50 mL , eyal I - of the
actual volume in the sample tube.
i. Make sure that the sample introduction tube is placed in the 2%
HCI rinse solution for at least 2-3 seconds t ~ n Sdl " 'e s
Analyze the :,landd, d~. and the Sdl l le s in the following order:
(1) Analyze the i"le""ediaLe check sLdl-ddld solutions.
(2) Analyze the ",eage,~l blank" (or sample blank co, ' ~ ,s
cobalt). The i"LensiLies of the analytes in this blank will be
suLLIduLed auLu",_'; 'Iy from the illLensiLies found in the
Sdl l l 'e
(3) Analyze each sample in d~
j. Results can be lepo,Lt:d in any convenient concer,L,dLiun units,
dependi"g upon how the tasks are p, uyl dl "" ,ed. Note: in
cases in which the analyst has entered the e-' b.dLion ~Landd,d~
into the task as "mg/L" and has entered the dilution volume as
"50 mL" and the sample weight in grams the sample results will
be in units of ,ug/g. The actual printout will show whatever units
are plUy~dl "",ed into the task for each element. The unit "~ug/g"
is prerer,ed to "ppm" for, epOI Lil ,9 sample results because the
latter term is dll b: _ous In labordLo~ ies where sample results
for this method are cor"monly, ~ o, led as "ppm" it must be
~" ,der:,Luod that this really means " ~ Uyl al l 15 of analyte per
gram of sample."
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A-3 VITAMIN C (L-ASCORBIC ACID) DETERMINATION
A. SAMPLE SIZE AND PRODUCT APPLICABILITY
Samples should be as uniform and ~I:pr~senldli~re of the product as
pc ~ ~- !e . Sampling should be pe, r~""ed i"""edi..~ly after a gentle mixing
or stirring to prevent inaccurate Sdll ,9 due to sl,~ rical;oll~ All sample
weights must be ,t:cGrded to at least three siy"ir,canl figures.
Sample sizes for low pH beverages are ~'~t~d from the ~ ri,.g
equation.
Sample size = 350/E
where:
Sample Size is the II ,eor~ al sample size, in grams; E is the ~ -e~--d
ascorL-.~ acid conce"l,dlion in ", _ dl"s per liter or h-__ dl" ,~spe- ti~ely,
as is and; 350 is the desired amount, in " Uy,d",~ (mcg), of ascorL:
acid in the sample prepd,dlion. The net conversion factor for ", oy~"~
to " _ dlll~ and h'-_ dlll:~ to grams is unity.
B. THEORY
In this method the amount of L-ascG,L:~ acid present in the sample is
dett:", ,ed by co- ~ ~ "~I, ic titration. A coulo" ,~I, ic method of analysis
measures the quantity of ele~;t, i~ ity required to carry out a cl ,e"
reaction. If the reaction is 100% efficient the p~cs~ge of one Faraday of
ele :L, i.;ily will cause the reaction of one equivalent weight.
In this case iodine is coulo",~l, 'Iy gene,dL~d from iodide. The iodine
then oxidizes the L-ascG,L ~ acid to dehy.l,uasco, ~ acid. When enough
iodine has been produced to oxidize all the L-ascorL ~ acid in the sample,
an excess of iodine will occur. This excess of iodine signals the
equivalence point, and is del~c1~d by two con~ld"L polenLidl ele~,udes.
The quantity of ele~, i~iily used is given by the product of current times the
time to reach the end point of the co~ Ldc titration. Thus the amount of
iodine used is equal to the number of equivalents of L-ascorL:- acid and
the amount of L-asco, L acid can be ~
Trichlor~,acelic acid is added to the sample to plec;~iLdl~: the protein and to
., ,c,;~, , the acidic con n necessal y for a quantitative reaction.
C. APPARATUS
Analytical Balance
~ Beakers 100ml, graduated
~ B-i"h",al1l1 E585 Polarizer
Cable for Double Platinum Wire Electrode, Brinkmann cat.
no. 20-97-738-8 or equivalent
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~ Cabies for Platinum Foil Cle~ udes, B~i"h~"dnn cat. no. 20-
97-770-1 and 20-00-853-9, or equivalent
~ Chart Paper
~ Dc~
~ Diâ,~:~,~ ' '3 Pipets
~ Disr ~ tips for pipettor
~ Double Platinum Wue E lecL,ude, Blillh.,'dnl1 cat. no. 20-92-
3504, or equivalent
~ CleuLI ude Holder
~ [ppendo, r pipet or equivalent, 200 mcl
~ Isolation Tube, outer iidr"~ler 20 mm, 125 mm long, Pore
Size C, Ace Glass Company cat. no. 7209-16; OR outer
d;dlllt:L~I 12mm, 125 mm long, Pore Size E, Ace cat. no.
7209-10. Size of isolation tube dependa on size of
ele~,L,udes used.
~ Keithly Model 225 Constant Current Source OR Keithly
Model 220 Constant Current Source
~ Magnetic Stir Plate
~ Pipettor; 5 ml - OxFord, Wheaton, CppendG, r, or
Fil " , p _:
Pipettor or dis"ense" 10 ml and 30 ml - Oxford, Wi ,edl~on,
Lab Industries or equivalent
~ Platinum Foil E Ic~,L,udes, (2), Bril,hlllann cat. no. 20-92-
110-2, orequivalent
~ Sample vials, 5 ml with screw caps
~ Shields: yellow or clear shields with a cutoff of 385
nanc" I I~L~I ~
Strip Chart Recorder; Kipp & Zonen or equivalent
~ Teflon-coated Stir Bars
UlLldsoh;~, Bath
Vacuum Flask, 2,000 mL
Volumetric flasks, 100, 500 ml, 1000 ml with :,Luuper:,
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D. REAGENTS
1. CHEMICALS
L( I ) asco,L acid (USP Rt:r~r~nce Standard, Official Lot); store
in a de~ r
M_Lapho:,~.horic acid; ACS or equivalent
Potassium iodide; ACS or equivalent
Sodium sulfate, anhydrous granular; ACS or equivalent
Trich!cruacc:Lic acid; ACS or equivalent
Il. SOLUTIONS
NOTE: All solutions, sa",, '-s and ~Ldnddld:~
must be pr~pa,ed and stored under UV shielded
or yellow sh- - 'c' o d lighting unless otherwise
stated (see APPARATUS).
NOTE: De ~sed water should be used for the
0.1M pot~csiurn iodide solution to prevent air
w~iddLiùl- of 1- to 12. Degas water by placing
deiolli,dd water into a vacuum flask, and
placing it under vacuum for 15 minutes with
sonicdliùn.
1. Poldasium lodide (0.1M)
Weigh 8.3 (~0.5) 9 of pot~csi~rn iodide into a 500 ml
volumetric flask. Dissolve and dilute to volume with
deg~-cserl. deiol,i~ed water. Store in a tightly sLuppe,~d
brown bottle at room L~r"perdLure. Do not store for more
than one (1 ) week. Discard if solution acq~ s a yellow
tinge.
2. L-Ascorbic Acid Stal~la-J (2000 mg/L)
Store sLdllddld bottle in a d~icc~ r to prevent moisture
absor~,Lion. Weigh accurately 0.200 (+0.0005) 9 and
transfer quantitatively to a 100 ml volumetric flask.
Dissolve and dilute to volume with 3% metaphosphoric
acid. The sLdndal d can be made fresh just before use
each day, or can be stored in small vials in the freezer.
Standard stored in the freezer is good for two months.
3 . T. ich lor~acelie Acid (1 M)
Weigh 163 (+0.5) 9 of trichloruac~Lic acid into a 1 liter
volumetric flask. Add 500 ml deg~-csed deior,i,ed water.
Swirl until di~solved, then dilute to volume. This reagent
~ may be stored for one month at room l~r"perdL.Ire.
4. Sodium Sulfate (1M)
Weight 142 (+0.5) 9 of sodium sulfate into a 1 liter
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volumetric flask. Add app,u~i,"aLely 750 ml of d~ioni~ed
water and mix until di~solv~d. Dilute to volume with
deio~ ed water. This reagent'may be stored for six
months at room len"~erdLure.
5. ~ t ,rhosph~r;. Acid (3%)
Accurately weigh 15.0 (iO.10) 9 of ",~Ld,i~l-ospl-oric acid
and quantitatively transfer to a 500 ml volumetric flask.
Add approxi",~t~ ly 250 ml deioni~ed water and swirl until
",~Ld~.ho~,horic acid is .li~.solved. Dilute to volume with
water. This solution may be stored for one week under
, ~r, i~e, dLi~ (2-8 ~C).
E. PROCEDURE
Instrument Settings - Figures 8 ' 10 and 11.
a. Recorder Chartspeed/input 1 mmlsecond or5
cm/min deper, ,9 on
chart~eco,der/l volt
b. Poldli~er Constant puL~:nLial 150 mV
E585 sensitivity 10 ", Udlll~J~
c. Keithly 1.56 ma
Constant
Current Source
1. Fill the isolation tube co" , ,9 the cathode 01e~ L, ude with
1 M sodium sulfate. The sodium sulfate continuously moves
from the isolation tube through the glass frit into the sample
solution and must be frequently r~ I n;sl,ed.
2. An instrument check should be done at the start of each day.
Using an [ppendo, r pipet (or equivalent) pipet 200
r" . li~ :, of the 2000 mg/L L-asco, L ~ ~ acid :~Ldnddl d into a
100 ml beaker. Follow steps 5 through 12 of the procedure.
Follow steps 1 through 4 of the ~'qtions. Compare the
ê~,uel il l l~l, 'Iy deLt:", ,ed conce, ILI dLion to the Ll ,eor~Lic al
concer,L,dLion. The results should be within 4% of the
e~ d value. If not run another instrument check using
fresh ,~ager,La and :,Ldnddld. If the e,~l,eri",er,Ldl result still
differs by more than 4% from the ex,ueuLed :~Ldnddld value
consult the method supervisor.
3. Agitate Sdll es well before and during Sdll ,9. Liquid
Sdl, - s must be freshly opened. Analyses must be
cGr, ~ t~ d within 20 minutes after the co" ~ ,er is opened.
Samples are weighed directly into the beaker unless
otherwise stated.
4. vVhile swirling the sample add 5.0 (+0.1) ml of 1M
Ll i.:hlo, oact:Lic acid (TCA) to the sample. Swirl for 30
seconds to cor" !~- 'y pl~u;~JiLdl~ the protein.
5. Add 30.0 (+0.5) ml of 0.1 M pof-qcsi- Im iodide to the sample.
6. Add deg~-csed dEioni~ed water to apl ruxi" Idt~ly the 60 ml
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mark.
7. Add a stir bar. Lower the ele_t,udes into the sample soiution.
Make sure that all the ele~;t, udes are i" " "er:,ed. The sodium
sulfate level in the isolation tube must be at least 2 cm above
the sample level. Adjust the " Id~l ,etic stirrer speed so that
stirring is vigorous but no air is entrained.
8. Switch the chart r~:corder on. Switch the poldl i~er on. Adjust
the base line of the chart ,~co"~er to 10% of full scale.
9. Switch the con~ld"L current source on.
10. Titrate until excess iodine is produced i" ~ by a rising
current curve. Stop the titration when the rising current curve
has reached at least 70% of full scale on the chart rt:corder
paper.
~ 11. Switch the r~corder chart speed to off and the con~LdnL
current source to standby.
12. Remove the ele~t,udes from the sample and rinse well with
deior,i~ed water.
F. CALCULATIONS
1. ExLIdpoldL~ the linear portion of the rising current curve to the
base line to locate the end point. The portion of the curve
b~:t~r _en 70% and 30% of full scale will be linear. (Figure 12).
2. Count the number of ce"Li",~t~r:. from start of titration to the
end point to the nearest 0.1 cer,Li",~ r.
3. Convert this di~Ad,)ce to seconds of titration time.
4. C ~ ~ the amount of L-asco, L ~ - acid present in the
sample by the ~ ~.; ,9 formula:
C=mxixt = ixtxR
nxsxF s
Vvhere:
C = concellL,dLion of L-ascorL: acid in mg/l or mg/kg
(mcg/ml = mg/l and mcg/g = mg/Kg)
m = 176 (gram ."~ weight of L-asco,L~- acid)
n = 2 (change in valence)
current in ~" IIIJa
t = time in seconds
F = 96487co~- "L,s/equivalent
R = prupo, Lion ~y consLa"L for m n and F=0.912 mcg
mA-sec
s = sample size in g
EXAMPLE: Assume a 3.0 9 sample of a low pH beverage was
analyzed and the measured length of titration on the
strip chart was 19.0 cm. Chart speed was 1 mm/sec.
The current during the titration was 1.56 mA
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s
where
i= 1.56mA; s= 3.0 9; R= 0.912 mcg/mA-sec; and
t= 19 cm x 1 sec x 1Omm = 190 sec
1 mm 1 cm
C = (1.56mA) x (0.912 mcg/mA-sec) x (190 sec)
3.09
= 90 mcg/g
= 90 mg/Kg
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