United States Patent .0. 71 .

2,914,446

sq ,Pefented Nov,- 24,195.91

1,
the present invention must have the following charac

2,914,446

teristics:

1.,

(1) Contains a nitrite (ONO) moiety; - ' '

NITRITE STABILIZED ASCORBIC ACID-CYANO

COBALAMIN COMPOSITIONS

. 0,0005% by weight NO2;>. .

This invention relates to vitamin compositions stabi

moiety.

(2)_ Is su?iciently soluble to supply at least about

Lee H. Macdonald, Paw Paw Township, Van Buren

' County, Mich., assignor to The Upjohn Company, Kal
amazoo, Mich., a corporation of Michigan
No Drawing. Application June 26, 1958
.10
Serial No. 744,642
5 Claims. (Cl. 167-81)

lized against decomposition, and more particularly to

vitamin compositions containing vitamin B12 stabilized
by the pressure of a stabilizing agent containing a nitrite

15

(3) Is not toxic and does not exhibitphysiological

activity per dosage unit-when used in a concentration

su?icient to supply the required amount of nitrite.

Among the stabilizing agents possessing the foregoing
characteristics are the following: 'the alkali metal and

alkaline earth nitrites, e.g., sodium- nitrite, potassium.

nitrite, magnesium nitrite, and calcium nitrite; the alkyl

nitrites, e.g., ethyl nitrite, amyl nitrite, octyl nitrite, propyl

nitrite, and butyl nitrite; acid nitrites, e.g., nitrous acid.
Because of its superior ability to ful?llthe'foregoing
requirements and having the additional advantage ofv

being economical, easily available, and convenience of

use, sodium nitrite is preferred.

In general, the various compositions of the present

Vitamin B12, or cyanocobalamin as it is named in 20 invention contain from about 0.0005 percent to about
The United States Pharmacopeia XV, is now ya recog
0.41 percent, by weight, of N02. For any particular
nized nutritional factor and requisite for normal blood
composition the amount of stabilizing agent to be used
formation. It is a speci?c treatment for certain anemias
is determined by (a) the particular nitrite used, and
and may be administered parenterally in solution or
(b) the concentration of cyanocobalamin. For example,

orally in tablet or liquid forms. In addition to compo 25 when sodium nitrite is used the amount would range
s'itions containing cyanocobalamin as the sole thera
from about 0.00075 percent to about 0.15 percent, by

peutic ingredient it is desirable that cyanocobalamin be

Weight, said amounts supplying the desired 0.0005 to
incorporated in a composition containing a plurality of
0.1 percent of N02. Amyl nitrite from about 0.00127_
other vitamins for prophylactic or therapeutic, treat
to 0.255 percent or nitrous acid from about 0.0005 to
ment of multiple vitamin de?ciencies. The preparation 30 0.1 percent can also supply the desired concentration of
of such a multiple vitamin composition confronts the
N02. For the other agents, the amount to be used is
art with the problem of reconciling the chemical incom
easily calculated from their molecular weights. On the
patibility between cyanocobalamin and ascorbic acid.
basis of a cyanocobalamin concentration of from about
The art, prior to the present invention, has attempted
one to about ten micrograms per milliliter, about 0.005.
to solve'this problem and several partially satisfactory 35 percent of sodium nitrite (equivalent to about 0.0033,
compositions have been announced. These compositions
percent N02) is preferred. Compositions containing an
are: a sugar coated tablet wherein one vitamin is in

corporated in the coating material and the other is placed

in the core of the tablet; a. liquid dispersion containing,

increased amount of cyanocobalamin would contain a

proportional increase in amount of stabilizing agent.

For example, a concentration of about 1000 micrograms
in addition to the vitamins, a water-swellable montmoril 40 of cyanocobalamin per milliliter would contain about:
lonite clay; and a solution wherein the cyanocobalamin
0.1 percent of sodium nitrite.
.
is converted to a cobalamin analogue by the addition
In general, solutions would contain the stabilizing
of sodium bisul?te. It is "apparent that of these com
agent dissolved in the solvent,_or in the case'of a vitamin,
positions the ?rst solves the problem only as to a solid
emulsion the stabilizing agent would be dissolved in the
oral form of medication; the second only as to an orally 45 aqueous phase. Tabletformulations containing cyano
administered medication, and the third does not solve ' cobalamin and ascorbic acid can be prepared ,andinade

the problem of stabilizing cyanocobalamin in the pres

more stable by dissolvingthe nitrite and cyanocobalamin

ence of ascorbic acid, but instead presents a composition

in the granulating ?uid.

of 'a'cobalamin'analogue and ascorbic acid.

'

The stabilizing agent can be effectively employed-in

It is therefore an object of the present invention to 50 soltuions in which the solvent system is partially or

provide a stable multiple vitamin composition contain

ing cyanocobalamin and ascorbic acid. A further object

_ predominantly organic, so long as the solvent system re-

mains capable of dissolvingthe stabilizer. .For example,

is to provide a process of stabilizing cyanocobalamin to

sodium nitrite can be used when glycerin, propylene
ascorbic acid. in vitamin compositions in liquid or solid
glycol, mannitol, sorbitol, glucose, sucrose, and other
form. A still further object is to provide a cyano 55 sugars or polyols are part of the solvent system. jItlwas
cobalamin stabilizing agent suitable for use in vitamin
unexpectedly found that the stability of cyanocobalamin
compositions for oral or parenteral use. Other objects
inithe presence of nitrite and ascorbic acid was enhanced
will be apparent to one skilled in the art to which this
when glycerin, in a concentrationfof about sixty per
invention pertains.
~
cent or more, or'propylene glycol, in a concentration
"The foregoing and additional objects have been ac; 60 of about ?fty percent or more, was a part of the set:

complished by the provision of a useful vitamin compo

sition comprising cyanocobalamin, ascorbic acid, and a
non-toxic stabilizing agent capable of providing a nitrite

vent system.

It was found that the pH of the solution modi?ed the

degree to which nitrite was effective in stabilizing cyano
moiety.

v
cobalamin in the presence of ascorbic acid. At a pH
The stabilizing agents used in the compositions of 6.5 of about 3 to about 3.5 the stabilization was maximal;

2,914,446 _

Decreasing stability was observed as the pH was adjusted

in either direction.

'

'

pared with the sodium nitrite being omitted from the

formula. This composition then had about 3.5 micro
grams/cc. of cyanocobalamin added and the composi
tion divided into four samples. One of the samples had

Aqueous multiple vitamin compositions of the present

invention are prepared by dissolving cyanocobalamin,
ascorbic acid, and the stabilizing agent in water. The

sodium nitrite in a concentration of about 0.005 percent

added. Another sample had about 0.05 percent of amyl

nitrite added. The two remaining samples had no stabi

solution can also have dissolved therein other vitamins

of the water soluble group.

It is undesirable to have the cyanocobalamin and

lizer added and served as controls. Three of the samples
ascorbic acid dissolved in the solvent before the addi
were heated at 56 degrees centigrade for 96 hours, and
tion of the stabilizing agent, therefore it is a preferred 10 the remaining control sample was cooled to ?ve degrees
procedure to either dissolve-the cyanocobalamin and
centigrade during the heating period. All samples were
the stabilizing agent in one portion .of the Water and the
then assayed for cyanocobalaminactivity by microbiologi
ascorbic acid in another portion and then to mix the two
cal method. > The results are tabulated in Table II.

solutions together, or, to ?rst dissolve'the cyanocobalamin

and, stabilizing agent in the solvent before the addition 15
of~the ascorbic acid.

TABLE II

- Whenthe composition is to take the form of a multiple

vitamin emulsion, the equeous phase of the emulsion is

Vitamin Bu

Sample

prepared by the procedure followed in preparing the

aqueous solutions. The fat soluble vitamins are dis
solved in a vegetable oil and a suitable emulsifying agent
added. The'oil and aqueous solutions are mixed together
and-stirred until an emulsionis formed.
The pH of the aqueous solution or aqueous phase of
the emulsion is adjusted to within the range of about 25

pH 3 to about 3.5 by the addition of either hydrochloric

acideor-sodium hydroxide. Such adjustment takes place

Activity
mcgJce.

Amyl nitrite, 0.05 percent _________ ..

Control Sample (Heated) _________ -_

2. 6
1. 6

_ Sodium nitrite, 0.005 percent.

Control Sample (Not Heated) _______ _-

3. 2
3. 5

It appears that although amyl nitrite is e?ective in

stabilizing cyanocobalamin in the presence of ascorbic
acid, it is less so than sodium nitrite.

as the ?nal step of the process. In many cases the pH

will-not have to be adjusted as the concentration of acidic

The mechanism whereby the cyanocobalamin is sta

vitamins, primarily the ascorbic acid, will give the com 30 bilized in the presence of ascorbic acid by nitrite is not
known. However, it is known that it is not a metathetic
position a pH within the desired range. Hydrochloric
mechanism whereby the cyanide moiety is replaced by
acid is added to a composition having a pH larger than
the nitrite ion resulting in the formation of a cobalamin
3.5 and sodium hydroxide is added when the pH is less
than 3.

analogue.

When, in the preparation of multivitamin tablets, it 35

In view of the report by Kaczka et al., in the J. Am.

Chem. Soc. 73, 3569 (1951), describing the preparation

of nitro-cobalamin by reaction of hydroxy-cobalamin with

is necessary to use a granulating ?uid which is a solvent

for ascorbic acid and cyaiiocobalamin, the cyanocobala

sodium nitrite and acetic acid, it seemed possible that

some analogous displacement of the cyanide moiety of
granulating-?uid. The tablet granulation is then formed
by the addition of the granulating ?uid to the powder 40 cyanocobalamin would take place upon the addition of
sodium nitrite to an acidic solution of cyanocobalarnin.
mixture containing ascorbic acid and the other vitamins.
min and nitrite stabilizing agent are dissolved in the

A spectrophotometric study was undertaken to determine

A vitamin composition was prepared as described in

Example 1 except that the sodium nitrite was omitted and

Whether any such analogous displacement does take place.

cyanocobalamin was increased to a concentration of two

The procedure used was as follows: two solutions of

composition as shown in Table III were prepared in

micrograms per cubic centimeter. The composition was
divided into eleven samples, two samples serving as con 45 stoppered ?asks and placed in an oil bath with an even
temperature of seventy degrees centigrade; aliquots were
trols and nine samples having various agents added. The
withdrawn at intervals as indicated in Table III and the
nine samples with the added ingredients and one control
absorbance measured using a Cary Model 14 spectro
were heated at 98 degrees centigrade for three and three
photometer. The results are tabulated in Table III.
fourths hours, then all samples (including the unheated
control) were assayed by the microbiological method de 50
scribed in The United States Pharmacopeia XV. The
TABLE III
results are shown in TableI.

Sample A contains 0.000028 molar cyanocobalamin

TABLE I

Sample

Agent Added

55 and 0.0095 molar sodium nitrite in aqueous acetic acid

with the pH of the solution being 3.5.

Sample B contains 0.0000365 molar cyanocobalamin

in aqueous acetic acid with the pH of the solution

Assay
Result,

vigmm

being 3.25.

u,

mgJec.

'

60
Sample A

none, Control, unheated ______________________ __

Sample B

Time

none, Control, heated ______ __

A361 ab-

Biotin, 0.1 mg./ec., heated _________ __

sorhittvtty

, Sodium Metabisul?te, 2 mg.lec., heat

A361 ab

sorbitivity

Sodium Nitrite, 2 mg.lcc., heated .... __

Sodium Nitrate, 2 mg./cc., heated _____________ __

Wild Cherry Fluid Extract, NF. 2%, heated-.

17. 51
17. 37
17. 77
17. 98
17. 75

Veegum R \,1.5%, heated ______________________ __

Sodium Cyanide, 5 mg./cc., heated.~

. Cobalt Chloride, 5 mg./ec., heated ____ __

Protein Hydrolysate, 10 mg./cc., heated__

V 1 Vecgum is the trede'name of the R. T. Vanderbilt Co. for a hydrated

letrzlmplex calcium, magnesium, aluminum silicate prepared from benton

70

17. 93
17. 91
17. 89
17. 80
17.61

In order to compare the stabilizing etiect of an or

ganic nitrite with an inorganic nitrite, a vitamin com

It is thus evident, from the spectrophotometric study

reported above, that no displacement of the cyanide
moiety of'cyanocobalamin by nitrite ion occurs.
The following examples are given by way of illustra

position similar to that describedin Example 1 was pre

tion only and are not to be construed as limiting.

2,914,446

T Example]

' Example 3

1000 cc. of the present invention'are prepared

1000 cc. of the composition of the present invention

are prepared from the following types and amounts of

a...

the following types and amounts of ingredients:

ingredients:

' -

Nitrous acid _________________ __v____ __gram? 0.05

Cyanocobalamin

Sodium nitrite

mg

50

Vitamin A palmitate __________________ __gm__.

Vitamin D3

'

Corn oil
Ribo?avin

5.55

mg

.42

gm
gm

16.66

Pyridoxine hydrochloride ______________ __gm_..

Glycerin

cc

Deionized water, q.s. ad ________ __'_____ __cc__

Phenol

3.5

> > v5,0

1
3

do____

4.0

q.s.

m _

1000

Boil about 1200 cc. of water for injection for ten min

0.5
'
_

'

utes with nitrogen gas passing through it. Cool the

water rapidly to about twenty degrees Centigrade under

l1

Saccharin sodium ____________________ __gm__..

Propylene glycol

' '

do

-Water for injection (air free by nitrogen),

83.33
1.66

Emulsifying agent ____________________ __gm__

gm
gm-

, 0.005

~ Thiamine hydrochloride _____________ __do_r____ 12.5

1.66 15

gm
mg

Preservative
Flavor

Nicotinamide

gm

Ascorbic acid
Cyanocobalamin

do
7 do

6.4 10 Pyridoxine hydrochloride ____'__.__-v._,____do___~_ ;_ 1.5

1.66
d-Pantothenyl alcohol _______________ __do____
15
1.66
Ascorbic acid
do
75

Thiamine hydrochloride ________________ ....gm-Pantothenyl alcohol __________________ __gm-_..

Nicotinamide

Ribo?avin

an atmosphere of nitrogen. In about 9000 cc. of the

20

water, dissolve the above ingredients in the order named.

Stir until dissolved, keeping the solution air free by means
of a nitrogen atmosphere. Filter with a suitable glass
?lter of ultra ?ne porosity into suitable glass containers.

25

Example 4

600

40
1000

The ribo?avin, thiamine hydrochloride, pantothenyl al

cohol, nicotinamide, pyridoxine hydrochloride, and sac

1000 tablets of the composition of the present invention

are prepared from the following types and amounts of

charin sodium are dissolved in 240 cc. of deionized

ingredients:
water. The cyanocobalamin and sodium nitrite are dis
solved in ten cubic centimeters of water and added to 30 Thiamine hydrochloride _____________ __grams__
the ?rst solution. The preservatives and ?avors are dis
solved in the propylene glycol and glycerin and added

12.5

Ribo?avin _________________________ __do__.._

10.5

Pyridoxine hydrochloride ____________ __do.._.._

2.2

Sodium nitrite ______________________ __do_.__..

Calcium stearate powder _____________ __do_..__
Sucrose (50% w./v.) aqueous solution ____ __cc__

0.05
35
25

to the aqueous solution. The vitamin A palmitate, vita

Dextro calcium pantothenate _________ __do____
24
min D3 and emulsifying agent are mixed with the corn
Nicotinamide
do ___
105
oil and added _to the aqueous solution. Additional de 35
Folic acid
do....__v 1.75
ionized water is added to make 1000 cubic centimeters
Ascorbic acid ______________________ __do-___ 150
and the mixture stirred until a uniform emulsion results.
Cyanocobalamin ____________________ __do_..-_ 0.005

Example 2
40

1000 cc. of the composition of the present invention

are prepared from the following types and amounts of in

The cyanocobalaminand sodium nitrite are dissolved in

gredients:

the sucrose solution. The remaining vitamin powders

are mixed together and a granulation formed by adding

Thiamine hydrochloride ____________ __gram__

Ribo?avin

do___-

Pantothenyl alcohol ________________ _._do____.

0.2 45 the sucrose solution, mixing, forcing through a screen,

and drying. The calcium stearate is mixed with the dry

granules and the mixture pressed into tablets.

0.2

0.5

Nicotinamide ______________________ __do___..

1.0

Pyridoxine hydrochloride ___________ __do____

0.05

Folic acid ________________________ __do____

0.05 50

Ascorbic acid _______________ __' ____ __do____

10.0

Example 5
1000 cc. of the present invention are prepared'from the
following types and amounts of ingredients:

Cyanocobalamin ___________________ __do____ 0.0005

Sodium nitrite ______________________ __gram__
1
Ferrous gluconate _________________ __do_..__
6.0
Cyanocobalamin ____________________ -..do____
1
Ethyl nitrite ______________________ __>do____
0.05
do
75
Saccharin sodium __________________ __do__.__
0.1 55 Ascorbic acid
Deionized water, q.s. ad ________________ __cc__ 1000
Preservative
do ___
1.0

Flavor

do____

3.0

Sorbitol, 70% solution ______________ __cc__

Deionized water, q.s. ad ________________ __cc__

650
1000

Hydrochloric acid, q.s.

The sodium nitrite and cyanocobalamin are dissolved in

about 100 cc. of the water. In the remaining water dis
solve the ascorbic acid and mix the two solutions together.
60
It is to be understood that the invention is not to be
limited to the exact details of operation or exact com

The sorbitol (70%) solution is heated to seventy de

grees centigrade and the ferrous gluconate added and
stirred until dissolved. The solution is allowed to cool

positions shown and described, as obvious modi?cations

cohol, nicotinamide, pyridoxine hydrochloride, folic acid,

and ascorbic acid as therapeutic ingredients and a nitrite,

and equivalents will be apparent to one skilled in the art,

and the invention is therefore to be limited only by the

to room temperature and the ascorbic acid is added and 65 scope of the appended claims.
stirred until dissolved. The sodium nitrite, cyanocobala
What is claimed is:
,
min, thiamine hydrochloride, ribo?avin, pantothenyl al
1. A vitamin composition comprising cyanocobalamin
and saccharin sodium are added to 250 cc. of deionized
said nitrite being present in a concentration equivalent to
water and the mixture stirred until dissolved. The two 70 from about 0.0005 to about 0.1 percent by weight of N02

solutions are mixed together and the ?avor and preserva

tive added. Additional deionized water is added to make
up a volume of 1000 cc. The pH is adjusted to within
the range of 3 to 3.5 by the addition of hydrochloric
acid.

of said composition.
2. The composition of claim 1 having a pH of from
about 3 to about 3.5.

3. A vitamin composition comprising cyanocobalamin

76 and ascorbic acid as therapeutic ingredients and sodium

2,914,446

nitrite, said sodium nitrite beitig present in a concen

tration equivalent totfrom about 0.0005 to about 0.1 per

References Cited in the ?le of this patent

,

cent by yveight of N02. of said composition.

4. The composition of claim 3 having a pH of from
about
_- ,5; A3-to
vitamin
aboutcompositioncomprising
3.5. ~
an oil-in~water

emulsion having ~a~pH of from about 3- to about 3.5 and a

plurality-of ,oil- soluble and- water soluble vitamins, in

cluding cyauocobalamin and ascorbic acid, and about

0005 percent-of- sodium nitrite.

'

V '

698,624

FOREIGN PATENTS 7

Great Britain '__'_'___'____'__ Oct. 21, 1953

OTHER REFERENCES

Prier: Arch. of Biochem. and Biophysics, vol. 40, N0.

2, October 1952, pp. 474-476.

'

'

Stapert: Jour. of the Am. Pharmaceutical vAsso., vol.

10 43, No. 2, February 1954, p. 87.