Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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LIQUIDS AND FOODSTUFFS CONTAINING
BETA-HYDROXY-BETA-METHYLBUTYRATE (HMB) IN FREE ACID FORM
Field of the Invention
(00011 The invention relates generally to nutritional supplements
containing [3-1-1ydroxy-13-
methylbutyrate (HMB) and more specifically to nutritional supplements
containing HMB in the
free acid form.
-Backeround of the Invention
1-00021 HMB has been found to be useful within the context of a variety of
applications.
Specifically, in U.S. Patent No, 5,360,613 (Nissen), HMB is described as
useful for reducing
blood levels of total cholesterol and low-density lipoprotein cholesterol. In
U.S. Patent No.
5,348,979 (Nissen et al.), HMB is described as useful for promoting nitrogen
retention in
humans. U.S. Patent No. 5,028,440 (Nissen) discusses the usefulness of HMB to
increase lean
tissue development in animals. Also, in U.S. Patent No. (1,992,470 (Nissen),
HMB is described
as effective in enhancing the immune response of mammals. U.S. Patent No.
6,031,000 (Nissen
et al.) describes use of HMB and at least one amino acid to treat disease-
associated wasting. In
U.S. Patent No. 6,103,764, IIMB is described as increasing the aerobic
capacity of muscle of an
animal without a substantial increase in the mass of the muscle. In addition,
HMB has been
described as useful for improving a human's perception of his emotional state
in U.S. Patent No.
6,291,525.
100031 HMB has been shown to have positive effects on maintaining and
increasing lean
muscle mass in cancer cachex i.a and AIDS wasting. In addition, a positive
effect on muscle
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damage and the resulting inflammatory response caused by exercising which
leads to muscle
soreness, strength loss, and an increase in pro-inflammatory cytokines is seen
with use of HMB.
1-00041 It has previously been observed that HMB alone or in combination
with other amino
acids is an effective supplement for restoring muscle strength and function in
young athletes.
Further, it has been observed that HMB in combination with two amino acids,
arginine and
lysine, is effective in increasing muscle mass in elderly persons.
1-00051 It is common to supplement traditional nutritional sources with
nutritional
supplements. Nutritional supplements come in many forms; for the present
invention, the focus
is on nutritional supplements in a non-emulsified substantially clear liquid
form. These
substantially clear liquid supplements often include vitamins, carbohydrates,
soluble protein,
amino acids, minerals and other nutrients. It is desirable to include HMB in
these liquid
supplements because of the known benefits of consuming HMB.
1-00061 It may also be desirable to include HMB in other liquids, such as
water, sports
drinks, fruit juices, soft drinks, and other carbonated and noncarbonated
beverages.
1-00071 Simply adding CaHMB to these liquids, especially substantially
clear liquid products
containing additional calcium, results in an instable product. It is has been
observed that adding
CaHMB to these liquids results, over time, in clumping, coagulation,
separation and
sedimentation. This clumping, coagulation, separation and sedimentation likely
occurs as at
least some of CaHMB dissociates over time and the dissociated calcium and
dissociated HMB
interact with the soluble protein and/or other components (including vitamins,
salts, and
minerals) in the liquid resulting in the observed clumping, coagulation,
separation and
sedimentation. Thus, clear liquid supplements containing CaHMB have not been
shelf stable,
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especially when the product has to be subjected to high heat during standard
sterilization
processes.
1-00081 It has been observed that CaHMB does not react well with
commercially available
soluble-protein containing ready-to-drink products, as addition of CaHMB
caused changes in the
physical appearance, including sedimentation and clumping.
1110091 In some instances, however, clear liquid nutritional supplement
products having
shelf stability with CaHMB have been described. For example, U.S. Patent
Application
Publication No. 2011/0305799 describes a clear liquid beverage containing
CaHMB. This clear
liquid nutritional product has a pH in the range of 2.8 to 4.6, and it is
stated in the application
that within these pH ranges, the CaHMB remains associated and thus the
interaction between the
calcium and the protein in the formulation is minimized or avoided, resulting
in minimizing or
avoiding gelation, sedimentation and coagulation and providing a shelf-stable
product.
1-000101 CaHMB has historically been the preferred delivery form of HMB.
Previously,
numerous obstacles existed to both extensive testing and commercial
utilization of the free acid
form of HMB, and since it was thought there was no difference between the two
forms from a
pharmacokinetic perspective, the calcium salt was adopted as a commercial
source of HMB.
Until recently packaging and, in particular, distribution of dietary
supplements has been better
suited to handle nutrients in a powdered form and therefore the calcium salt
of HMB was widely
accepted. HMB-acid is a liquid and much more difficult to deliver or
incorporate into products.
1-000111 Currently, the manufacturing process for HMB has allowed for HMB
free acid to be
produced in a purity that allows for oral ingestion of the HMB free acid.
Besides having a
commercial source that is pure enough for oral ingestion, the HMB- acid needs
to be buffered for
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oral ingestion, a process which only recently was determined due to the
factors listed above
which precluded previous use of HMB-acid.
[000121 It was assumed that ingestion of CaHMB would result in a rather
quick dissociation
of HMB from the calcium salt form. However, a recent study and corresponding
patent
application (U.S. App. Publication No. 20120053240) has shown that HMB in the
free acid form
has rather unique pharmacokinetic effects when compared to CaHMB ingestion.
Use of HMB
free acid (also called HMB-acid) improves HMB availability to tissues and thus
provides a more
rapid and efficient method to get HMB to the tissues than administration of
CaHMB. It was
shown that in many instances HMB free acid is a better delivery form of HMB.
[000131 Thus, it is desirable to use the free acid form of HMB in place of
CaHMB in order to
take advantage of free acid HMB's unique pharmacokinetic effects. It was
expected, however,
that addition of HMB-acid to a non-emulsified liquid nutritional supplement
would have similar
results to the results observed when CaHMB is added to a liquid. Specifically,
it was expected
that addition of HMB-acid would result in a layer of HMB-acid appearing at the
top of the liquid,
that the HMB acid would be observed floating through the liquid and/or that
the HMB-acid
would interact with the other ingredients in the liquid causing clumping,
sedimentation and
changes to the physical appearance of the beverage. It was thus surprising and
unexpected that
the addition of HMB-acid to a liquid nutritional supplement resulted in the
HMB-acid
disappearing almost immediately and remaining in solution indefinitely.
[000141 Thus, a need exists for beverages and non-emulsified substantially
clear liquid
nutritional supplements containing HMB in the free acid form.
Summary of the Invention
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[000151 In one embodiment. the present invention is a liquid nutritional
supplement
containing HMB in the free acid form.
[000161 In another embodiment, the present invention is a clear liquid
beverage containing
HMB in the free acid form.
[000171 The nutritional supplement or beverage may contain calcium and
soluble protein.
[000181 In yet another embodiment, the present invention is a method of
adding free acid
HMB to a liquid nutritional supplement Or beverage. The addition of free acid
HMB may occur
during the process of manufacturing a liquid or beverage product or it may be
done immediately
prior to consuming the liquid or beverage.
[000191 Surprisingly, even though it was thought that free acid HMB and
calcium (if present)
in a clear liquid nutritional supplement containing soluble protein was not
shelf stable and would
result in the HMB and/or calcium (if present) interacting with the soluble
protein to cause
sedimentation and separation, the present invention comprises such a
nutritional supplement
containing soluble protein and free acid HMB and is still shelf stable. In
addition, the addition of
free acid HMB to a liquid that does not contain soluble protein also
surprisingly does not result
in a liquid sedimentation and separation that would be expected upon the
addition of free acid
HMB.
Brief Description of the Figures
Fig. 1 shows photographs of the bottles showing the physical appearance of the
liquid
supplements studied in Example 1 at days 1, 4, 14 and 21.
Fig. 2a shows photographs of the bottles of ISOPURE showing the physical
appearance
of the IS OPURE samples studied in Example 3 at days 0,7, 14,28 and 42.
Fig. 2b shows photographs of the bottles of GNC-Protein Juice showing the
physical
appearance of the GNC-Protein Juice samples studied in Example 3 at days 0, 7,
14, 28 and 42.
Fig. 2c shows photographs of the bottles of Gatorade Recovery showing the
physical
appearance of the Gatorade Recovery samples studied in Example 3 at days 0, 7,
14, 28 and 42.
Fig. 2d shows photographs of the bottles of Spartos Protein water showing the
physical
appearance of the Spartos Protein water samples studied in Example 3 at days
0, 7, 14, 28 and
42.
Fig. 3 is a graph depicting the stability results of 300mg of HMB-acid over
120 days.
Fig. 4 is a graph depicting the stability results of 1000mg of HMB -acid over
120 days.
Fig. 5 is a graph depicting the stability results of 3000mg of HMB -acid over
120 days.
Detailed Description of Preferred Embodiments
1000201 In the preferred embodiment, the invention is a non-emulsified,
substantially clear
liquid nutritional supplement, most typically a beverage or drink, containing
free acid HMB.
The liquids of the present invention may contain water and free acid HMB.
Additional
ingredients may include soluble protein, amino acids, carbohydrates, vitamins
and minerals such
as calcium.
1000211 The invention comprises, at its base, HMB-acid and a liquid. In
most instances, the
liquid has a crude fat content of less than 1%. One of skill in the art will
understand that the
invention may include free fatty acids, such as DHA or EPA, yet still be
considered to have less
than 1% crude fat content. Water, sports drinks, juices, soft drinks,
pharmaceutical liquids and
substantially clear nutritional supplements such as Ensure Clear, IsoPure0
protein drink,
GNCO protein juice, GatoradeTM Recover, and SpartosTM protein water may
comprise the liquid
of the present invention, although the invention is not limited to these
liquids.
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[000221 While the preferred embodiment is HMB-acid in a liquid nutritional
supplement, in
another embodiment, HMB-acid may be added to food, such as honey, applesauce,
fruit ribbons
and other foodstuffs in the manufacturing process or after manufacturing prior
to ingestion by the
consumer.
[000231 In the present invention, HMB-acid is present in an effective
amount. An effective
amount includes a range from about 0.01 grams to about 0.2 grams of HMB-acid
per kilogram
body weight in twenty-four (24) hours. HMB-acid may also be administered to a
human in an
effective amount from about 0.5 grams to about 30 grams of HMB-acid per day.
Thus, premade
products that include HMB-acid will have HMB-acid present substantially in
these ranges.
Individual servings of HMB-acid to be added to a liquid or foodstuff will also
have HMB-acid
present substantially within these ranges.
[000241 Any soluble protein source is suitable for use in the present
invention, including but
not limited to whey protein concentrate, whey protein isolate, casein
hydrolysate, hydrolyzed
collagen and combinations thereof.
[000251 The liquid nutritional supplement may also include calcium, most
preferably in a
soluble form to minimize interaction with other components of the supplement
(such as the free
acid HMB or soluble protein if present). Nonlimiting examples of appropriate
forms of calcium
include calcium carbonate, calcium citrate, calcium gluconate, calcium
lactate, calcium
phosphate and calcium pantothenate.
[000261 The liquid nutritional supplement of the present invention may also
include vitamins,
such as vitamin D. As described in U.S. Patent Application Publication No.
2010/0179112, the
combination of vitamin D and HMB has a synergistic effect, thus it may be
desired to include
vitamin D in the liquids of the present invention. Vitamin D may be included
in any form. and
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most preferably as Vitamin D3, also known as cholecalciferol. The amount of
vitamin D to be
included can be any amount of vitamin D, up to an amount causing toxicity.
Typical amounts of
vitamin D are up to 40001U, though much higher amounts may be included as
described in U.S.
Patent Application Publication No. 2010/0179112.
[000271 HMB-acid as used in this invention is generally in a liquid form.
Other forms of
HMB-acid within the scope of this invention include HMB-acid in a gel or gel-
matrix form. Any
and all forms of HMB-acid may be used in the present invention.
[000281 In the present invention, free acid HMB may be added to a liquid at
any point prior
to consumption. For example, free acid HMB may be added to a liquid or
foodstuff during the
manufacturing process and at any time during the manufacturing process.
Suitable methods of
manufacturing include but are not limited to a hot fill process, retort or
aseptic filling of liquid
products and the HMB-acid may be added at any time during any of these
manufacturing
processes.
[000291 There is no need to subject the liquid or foodstuff to any non-
standard manufacturing
steps, such as additional heating or cooling steps, or additional mixing or
creation of a slurry.
The HMB-acid can be added at any time during any manufacturing process and the
product will
be shelf-stable with minimal separation or sedimentation of the HMB-acid
interacting with other
components of the liquid or foodstuff.
[000301 HMB-acid may be added to a liquid or foodstuff after manufacture
and prior to
consumption. For example, HMB-acid may be provided separate from the liquid or
foodstuff for
a consumer to add to a liquid or foodstuff of their choice. Discretely
measured packages of
HMB-acid, most typically in a concentrate form, and most typically a gel form,
may be provided
to add to a defined amount of water, sports beverage, juice, substantially
clear nutritional liquid,
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etc. The HMB-acid may be provided in single or multiple dose packages. The HMB-
acid will
mix in any potable liquid or foodstuff and is soluble in the aforementioned
liquids and
foodstuffs.
[000311 The present invention is not dependent on the pH of the liquid base
to which HMB-
acid is added. The pH of the liquid base may be as low as about 2Ø
[000321 The observation in the present invention that the addition of HMB-
acid to liquids and
foodstuffs would result in liquids and foodstuffs with very little
sedimentation, gelation or
coagulation of the HMB-acid interacting with other components of the liquid or
foodstuff is
surprising and unexpected. It is known in the art that the addition of calcium
HMB to liquids
such as water or liquids containing soluble protein results in the partial
dissociation of calcium
and HMB into the ion forms, resulting in sedimentation, gelation and
coagulation. While U.S.
Patent Application Serial No. 2011/0305799 describes the addition of calcium
HMB to
substantially clear nutritional liquids, it is made clear the fact that the
calcium HMB remains
associated, thus avoiding the interaction between the calcium and protein
which minimizes these
undesired effects. HMB-acid is by definition HMB in a dissociated form and it
was expected
that the free acid form would interact with soluble proteins and other
components of the liquid
which would result in sedimentation, etc. Surprisingly, the free acid form of
HMB does not
interact with any of the other components, thus providing a stable product
with little to no
separation or sedimentation. The observation that HMB-acid goes into solution
almost instantly
when added to a liquid was also unexpected. It was expected that the addition
of HMB-acid to a
liquid would result in the HMB-acid forming a layer at the top of the liquid,
or floating
throughout the liquid or that the HMB-acid would interact with the other
ingredients in the liquid
causing clumping, sedimentation and changes to the physical appearance of the
beverage.
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Instead, addition of HMB-acid followed by slight shaking results in the
immediate disappearance
of the HMB-acid and a crystal clear liquid.
[000331 Further, the addition of HMB-acid had little to no impact on the
flavor, texture,
mouthfeel and stability of the liquid drink matrix.
[000341 It is understood by one of skill in the art that consumption of HMB-
acid marked to
be added to a liquid without adding the HMB-acid (usually in the form of a
gel), is included in
the scope of this invention.
EXPERIMENTAL EXAMPLES
Example 1: Stability of Product Composition with the addition of HMB (free
acid and
Calcium salt forms)
Protocol:
1. N = 2 (Two flavors of a commercially available ready-to-drink soluble-
protein product; Ensure
Clear, pH approximately 2.74)
2. Label the glass bottles A1-A3 and B1-133. The labels correspond to the
following conditions:
Table 1
Label Condition
Al Peach flavor control
A2 Peach flavor CaHMB
A3 Peach flavor HMB FA
B1 Blueberry pomegranate control
B2 Blueberry pomegranate CaHMB
B3 Blueberry pomegranate HMB FA
3. To bottles A2 and B2 lg of CaHMB MTI 1208-100-02 TSI 12070630 added.
4. To bottles A3 and B3 0.8g of HMB Free Acid MTI 1103-100-09 Lot 11010084
added.
5. 40m1 of Peach flavored product added to the bottles labeled A1-A3.
6. 40m1 of Blueberry Pomegranate flavored product added to the bottles labeled
B1-133.
7. Bottles were sealed with rubber caps and crimped shut. Then, they were
mixed well and placed
on shelf.
8. Appearances were noted and photos were taken daily (bottles were not
disturbed).
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Results:
Table 2 reports the physical appearance of the ready-to-drink soluble-protein
product (n=2)
following the addition of CaHMB or HMB free acid and mixing. HMB was not added
to the
control bottles. Day 1 was recorded after initial mixing.
Table 2
Control/No
HMB w/ CaHMB W/HMB free acid
Day 1 Semi-Clear Cloudy Semi-Clear
Day 7 Semi-Clear Defined Separation Semi-Clear
Day
14 Semi-Clear Defined Separation w/Particulates Semi-Clear
Day
21 Semi-Clear Defined Separation w/Particulates Semi-Clear
In addition to the physical appearance notes, photos were taken at the time
points noted above.
These photos appear in Fig 1.
Example 2:
Study: Taste of Product with the Addition of HMB (free acid and Ca salt forms)
Protocol:
1. N = 2 (Two flavors of a commercially available ready-to-drink soluble-
protein product; Ensure
Clear, pH approximately 2.74)
2. Label the glass bottles A1-A3 and B1-133. The labels correspond to the
following conditions:
Table 3
Label Condition
Al Peach flavor control
A2 Peach flavor CaHMB
A3 Peach flavor HMB FA
B1 Bluebeffy pomegranate control
B2 Blueberry pomegranate CaHMB
B3 Blueberry pomegranate HMB FA
3. To bottles A2 and B2 1g of CaHMB MTI 1208-100-02 TSI 12070630 was added and
mixed.
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4. To bottles A3 and B3 1.0 g of HMB Free Acid Lot 11010086 was added and
mixed.
5. Bottles were tasted by investigator.
6. Between each sample tasting, a water mouth-washout was performed and 1
minute wait.
Results:
Table 4 reports the taste of the ready-to-drink soluble-protein product (n=2)
following the
addition of CaHMB or HMB free acid and mixing. HMB was not added to the
control bottles.
Table 4
Control/No w/ CaHMB W/HMB free acid
HMB (1) (2) (3)
No change in
Slight change in flavor; flavor;
A Sweet/Sour ¨ metallic after-taste
Slight increase in
peach bad mouthfeel sour after-taste
Slight change in flavor; No change in
Sweet/Sour ¨ metallic after-taste flavor;
blueberry/porn bad mouthfeel Sour after-taste
Conclusion:
The soluble-protein ready-to-drink products containing HMB free acid were
surprisingly stable
compared to those containing CaHMB. Additionally, the addition of HMB free
acid had no
impact on the physical appearance, mouthfeel or texture of the product when
compared to the
control with only a slight change in after-taste (more sour which is likely
due to the low pH of
the HMB free acid).
Example 3
Lab Study #21 Long term stability
1. Rinse 12 glass bottles with ethanol and let air dry overnight.
2. Label the glass bottles A1-A3, B1-133, C1-C3 and D1-D3. The labels
correspond to the following
conditions:
Table 5
Label Condition
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Al 40m1
A2 250mg CaHMB in 40m1
A3 200mg HMB FA in 40m1
B1 40m1
B2 250mg CaHMB in 40m1
B3 200mg HMB FA in 40m1
Cl 40m1
C2 250mg CaHMB in 40m1
C3 200mg HMB FA in 40m1
D1 40m1
D2 250mg CaHMB in 40m1
D3 200mg HMB FA in 40m1
3. To bottles A2-D2 CaHMB MTI 1208-100-02 TS! 12070630 was used.
4. To bottles A3-D3 HMB Free Acid MTI 1103-100-09 Lot 11010084 was used.
5. Add 40m1 of ISOPURE protein drink to A1-A3, 40m1 of GNC protein juice to
B1-133, 40m1
Gatorade Recover to C1-C3, and 40m1Spartos protein water to D1-D3.
6. Flush with nitrogen and seal the bottles with rubber caps and metal clamp
caps.
7. Mix well.
8. Take photo of bottles A1-A3, B1-133, C1-C3, and D1-D3 every Friday. The
photos appear in Fig.
2a-2d.
The following tables 6-9 provide the nutritional information for each of the
liquids tested
(ISOPURE, GNC Protein Juice. Gatorade G3-Recover, and Spartos protein water.)
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Table 6
Nutrition Facts: Grape Frost ISOPURE¨Fig. 2a
Serving Size: 1 Bottle (20 fl. oz/591 mL)
AMOUNT PER DAILY VALUE
SERVING
Calories 160
Calories from Fat 0
Total Fat 0%
Sodium 80mq 3%
Potassium 45mq 1%
Total Carbohydrate 0%
Protein 40q 80%
Vitamin A 0%
Vitamin C 0%
Calcium 6%
Iron 0%
Table 7
GNC ¨ Protein Juice ¨ Mixed Berry¨Fig. 2b
Serving size 14 fl. Oz/414m1
Servings per container: 2
AMOUNT PER DAILY VALUE
SERVING
Calories 170
Calories from Fat 5
Total Fat _Qg 0%
Sodium 50mq 2%
Potassium OMQ 0%
Total Carbohydrate 26q 9%
Dietary Fiber 1g
24q
Protein 15q 30%
Vitamin A 10%
Vitamin E 10%
Niacin 10%
Vitamin B6 10%
Vitamin B12 10%
Pantothenic Acid 10%
Table 8
Gatorade G3 - Recover- Mixed Berry¨Fig. 2c
Serving size 8 fl. oz/240m1
Servings per container: 2
pH 3.68
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AMOUNT PER DAILY VALUE
SERVING
Calories 110
Calories from Fat 0
Total Fat 0%
Sodium 105mq 4%
Potassium 40mq 1%
Total Carbohydrate 20q 7%
Dietary Fiber
Sugars 20q
Protein 16%
Vitamin A 0%
Vitamin C 0%
Calcium 4%
Iron 0%
Table 9
Spartos protein water¨Fig. 2d
Serving size 8 fl. oz/240m1
Servings per container: 2
pH approximately 3.05
AMOUNT PER DAILY VALUE
SERVING
Calories 60
Calories from Fat 0
Total Fat 0%
Sodium 10mq <1%
Potassium 20mq 1%
Total Carbohydrate 3%
Protein 10%
Vitamin A 5%
Vitamin C 5%
Calcium 5%
Vitamin D 5%
Vitamin E 5%
Vitamin B6 15%
Folic Acid 15%
Vitamin B12 15%
Magnesium 5%
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As seen in Figs. 2a-2d, the liquids to which calcium HMB was added are cloudy
and contain
precipitate by 42 days. The calcium HMB bottles are bottles A2, B2, C2 and D2.
The bottles
containing liquid and HMB-acid (A3, B3, C3 and D3) are clear and precipitate-
free at 42 days
and look the same as the control bottles (no HMB) (Al. B1, CI and DI).
Example 4
This experiment was designed to observe the stability of HMB-acid at various
concentrations in soluble protein over 120 days.
To analyze the stability of HMB free acid in the soluble protein ( ISOPURE
protein
drink, Natures Best , Hauppauge. NY), different concentrations of HMB free
acid were added
to the recommended serving size of ISOPURE protein drink. The three
concentrations were
300mg/240m1, 1000m2/240m1. and 3000mg/240m1. HMB free acid was measured for
each
solution using HPLC at 5 different time points: 0, 30, 60, 90, and 120 d. HMB
free acid used
was MTI 1301-100-03 (Lot 120111036). ISOPURE protein drink used was Lot
1212111132310.
ISOPURE protein drink has a pH of approximately 2.54. All glassware and
supplies used were
autoclaved in preparation of the three different concentrations of HMB free
acid in ISOPURE
protein drink for the 5 different time points. Materials to be autoclaved were
as follows: glass
bottles, rubber and metal seals, pipette tips, and graduated cylinder.
Autoclave Method #1 for dry
goods in Building 3 of ISU Research Park was used.
Table 10 shows bottle labeling:
Table 10
Label Concentration( Time point
mg/ 240m1)
AO 300 0
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Al 300 30 days
A2 300 60 days
A3 300 90 days
A4 300 120 days
BO 1000 0
B1 1000 30 days
B2 1000 60 days
B3 1000 90 days
B4 1000 120 days
CO 3000 0
CI 3000 30 days
C2, 3000 60 days
C3 3000 90 days
C4 3000 120
ays
For bottles in group A, 60mg of HMBFA into each of the five bottles was added,
and then 48m1
of ISOPURE protein drink was added. The HMBFA concentration was 1.25mg/ml.
For bottles in group B 200mg of HMBFA into each of the five bottles was added,
and then 48m1
of ISOPURE protein drink was added. The HMBFA concentration was 4.16mg/ml.
For bottles in group C, 600mg of HMBFA into each of the five bottles was, and
then 48m1 of
ISOPURE protein drink was added. The HMBFA concentration was 12.5mg/ml.
ISOPURE protein drink has a protein concentration of 0.0677g/m1; the amount of
protein in each
sample was 3.25 g.
Table 11 shows actual weight(mg) and concentrations(mg/48m1) of samples.
Table 11
Label Weight (mg) Concentration
mg/ml)
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AO 72.6 1.51
Al 61.6 1.28
A2 67.8 1.41
A3 73.5 1.53
A4 65.3 1.36
BO 212.5 4.43
B1 213.6 4.45
B2 200.3 4.17
B3 206.1 4.29
B4 201.7 4.2
CO 614.4 12.8
Cl 612.8 12.77
C2 610.6 12.72
C3 613.1 12.77
C4 613.9 12.79
Each sample bottle was flushed with nitrogen and then the top of the bottle
was sealed
with rubber caps, crimped shut and mix well.
One bottle of each concentration was tested for IIMB using SOP 113-04 IIMB
(modified
standard curve), at Time 0, 30, 60, 90, and 120 d.
Table 12 shows the HMB standard curve to use.
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Table 12
HMB STD HPLC HMB Std .02M
20mg/mL Phosphate
Buffer
0 0 .75mL
0.5 25u1 .725mL
1.0 50u1 .700mL
2.0 100u1 .650mL
4.0 200u1 .550mL
8.0 400u1 .350mL
To prepare the samples for testing, 0.5m1 of the sample was transferred to a
microcentrifuge tube in quadruplet. Then 0.25m1 of KIV internal standard added
to standards and
three of each sample set. Finally, 0.25m1 0.2M phosphate buffer was added to
the three samples
with KIV internal standard and 0.5m1 0.2M phosphate buffer to the samples not
containing KIV.
The concentrations of each HMBFA sample were now: 0.625mg/ml, 2.08mg/ml, and
6.25mg/m1 respectively.
The samples were considered stable with a 5 % of expected change HMB
concentration
over the four months with an allowable 3% analytical variation. A linear
regression was
conducted on the data as a secondary measure of stability.
Results
The three prepared concentrations of HMB Free acid with ISOPURE sports drink
were
analyzed for HMB concentrations using HPLC at 0, 30, 60, 90 and 120 d. The
ingredients in the
ISOPURE sports drink interfered with OUT KIV internal standard peak so
external standard based
calculations were used to quantify HMB in each sample. The quantified amount
of HMB was
compared to the expected amount of HMB for each sample at each test time and
expressed in
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percent of expected. The results are presented in Figs.3-5. In general, the
three different
concentrations of HMB-FA in the sports drink tended to numerically decrease
over time but no
significant linear decrease was detected, p> 0.05. The changes were less than
8.0 %. The 300,
1000, and 3000 mg /serving of HMB-FA sample varied from 98.6% to 91.84, 98.1%
to 94.96,
and 97.28% to 93.57%% of expected, respectively.
Fig 3 shows samples containing 300mg of HMB free acid/serving of soluble
protein that
were evaluated for stability for 120 days. The decrease in HMB free acid was
less than 8% of
the expected amount of HMB free acid and there was no significant linear
decrease, p<0.05.
Fig. 4 shows samples containing 1000mg of HMB free acid/serving of soluble
protein
that were evaluated for stability for 120 days. The decrease in HMB free acid
was less than 8%
of the expected amount of HMB free acid and there was no significant linear
decrease, p<0.05.
Fig. 5 shows samples containing 3000mg of HMB free acid/serving of soluble
protein
that were evaluated for stability for 120 days. The decrease in HMB free acid
was less than 8%
of the expected amount of HMB free acid and there was no significant linear
decrease, p<0.05.
This experimental example demonstrates that the addition of HMB-acid to a
drink
containing soluble protein results in HMB-acid remaining stable at room
temperature for 120
days.
