oral multivitamin
stability study of
M any oral multivitamin drop liquid
preparations on the market often
have been prescribed or recommended
by physicians, especially in pediatric
and geriatric practice. Although they
enjoy considerable popularity, no of-
ficial recognition has been given to
them and therefore no official standards
have been established.
Early workers such as Stone! and
Campbel12 reported that many multi-
vitamin preparations tend to lose their
potency during the ordinary shelf-life
period and, for that reason, Delgado,
Lofgren and Burlage 3 and Parikh
and Lofgren 4 attempted to formulate
preparations which were more stable
than those on the market. Both
Delgado and Parikh found that if a
vehicle could be found which was a
solvent for the multivitamins and that
its water content could be reduced to a
minimum, the stability of the prepara-
tion could be increased.
With this factor in mind the present
work was undertaken with a mixture of
polyethylene glycol 200 and propylene
glycol as the main vehicle and only
three percent of water was required to
give a clear solution.
experimental
The following concentration of vi-
tamins was used in this study since
it approximates the composition of
many commercial products- Vitamin
A, 5,000 units; Vitamin D, 1,000 units ;
Ascorbic acid, 50 mg ; Thiamine
hydrochloride, 1 mg; Riboflavin, 1
mg; Pantothenic acid, 5 mg; Pyri-
doxine hydrochloride, 1 mg; Vitamin
B 12, 4 mcg; Vehicle, q.s. to 0.6 ml.
To obtain a clear solution of all these
vitamins and to improve stability, we
made various additions to the for-
mulation and in some cases used another
form of the vitamin. The source of
vitamins At and Dt was an oil concen-
tration of each containing one million
units per gram. To "solubilize" these
concentrates, we had to add poly-
sorbate 80. Since traces of metal in
the formulation may cause rapid de-
terioration of ascorbic acid, disodium
calcium ethylenediaminotetracetate was
* Presented to the section on pharmaceutica l
technology at the annual meeting of the AMERICAN
PHARMACEUTICAL ASSOCIATION in Las Vegas ,
Nevada, March 29,1962.
t Vitamin A Palmitate, synthetic in corn oil,
Type NYQ P-2.
t Viosterol, one million units per gram , Stand-
ard Brands, Inc.
86 Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION
liquid preparation *
by Vishnu D. Gupta and Frederick V. Lofgren
added as a sequestering agent. Also a
mixture of nordihydroguiaretic acid
and butylated hydroxyanisole was added
as antioxidants and a mixture oimethyl
and propyl paraben as preservatives.
Instead of dissolving riboflavin, which
is of low solubility in the formulation,
we used an equivalent amount of
ribofla vin -5-phospha te monosodium h y-
drate with greater solubility. Panto-
thenic acid is very unstable in most
formulations; therefore the more stable
d-pantothenyl alcohol was used in
equivalent amounts.
A st'ries of experiments to dissolve
these vitamins in a vehicle containing
various mixtures of polyethylene glycol
V. D. Gupta was pharmaceutical
200 and propylene glycol showed that chemist in charge of quality con-
a mixture of three parts by volume of trol for Schlicksup Drug Company
polyethylene glycol 200 and one part of Peoria, Illinois, until he left in
of propylene glycol gave the clearest 1963 to visit his home in India. He
solution; for this reason the mixture earned his BS in 1953 and his MS in
1957 from Banjab University in
was selected for use in this study. India. Following his graduation he
The final formulation was- was employed in the pharmaceu-
tical industry of India for two
Thiamine hydrochloride 1.66 Gm years. He came to the United
Sodium bicarbonate 20.00 Gm States in 1960 and began studying
at the University of Texas. When
Ascorbic acid 83.00 Gm
D-Pantothenyl alcohol 7.14 Gm he received his MS degree in
1961, he entered indusrial pharm-
Nicotinamide 16.60 Gm acy in this country.
Pyridoxine hydrochloride 1.66 Gm
Riboflavin-5-phosphate
monosodium 2.374 Gm
Vitamin B12 (1,000 mcgl
Gm) 6.64 ml
Vitamin A oil concentrate
(1 million USP units AI
Gm) 8.30 Gm
Vitamin D oil concentrate
(1 million USP units DI
Gm) 1.66 Gm
Nordihydroguiaretic acid 0.996 Gm
Butylated hydroxyanisole 0.50 Gm
Polysorbate 80 80.00 ml
Disodium calcium ethyl-
enediaminetetracetate 0.10 Gm
Methylparaben 1.80 Gm
Propylparaben 0.20 Gm
Recently boiled and
cooled distilled water 30.00 ml
Mixture of polyethylene
glycol 200 and propyl-
ene glycol (3:1 vIv) q.s. F.V. Lofgren, professor of phar-
macy at the University of Texas,
to 996 ml
earned his BS, MS and PhD (1930)
The final formulation was made up at the University of Washington.
A professor of pharmacy at Val-
using sodium bicarbonate to partially paraiso University in Indiana for
neutralize the ascorbic acid bringing the ten years, he left to become scien-
pH of the solution to 3.8. This for- tific director of Hart Drug Corpora-
mulation was divided into four portions tion in Miami, Florida from 1940 to
1950 and then returned to educa-
of approximately 240 ml and stored in tion in manufacturing pharmacy
12-fluid-ounce, amber bottles. They at the University of Texas. Since
were designated as formulations one, 1920 when he joined APhA he has
two, three and four. served on various committees of
the association and is now a life
econti nued on next page ) member.
 Formulation one with a pH of 3.8 Table I
was stored in the dark at room temper-
ature (approximately 25 degrees C) stability values for the formulation #1, pH 3.8*
with the air above the solution being
replaced by nitrogen. Each time the Vitamin Percentage retention after
bottle was opened, nitrogen was again oday 30 days 60 days 90 days
introduced above the solution.
Formulation two with a pH of 3.8 Vitamin A 101.66 95.14 91.40 86.34
Ascorbic acid 99.80 95.23 92.92 91.01
was stored in the dark at 37 degrees C. Thiamine hydrochloride 100 .05 94.27 90 .37 89.26
Formulation three was adjusted to a Riboflavin 100 .44 98.84 97.47 96.83
pH of 2.8 by the addition of a few drops Pyridoxine hydro-
of diluted hydrochloric acid. It was chloride 98.20 98.21 98.20 97.92
Nicotinamide 98.16 97.97 96.77 94.27
stored in the dark at 37 degrees C. Vitamin B l2 100.13 88.05 80.23 72.88
Formulation four was adjusted to a
pH of 4.8 by the addition of a few drops * Formulation was incubated in the dark at room temperature in an amber colored
bottle in which the air was replaced by nitrogen.
of 10 percent sodium bicarbonate so-
lution . It was stored in the dark at
37 degrees C. Table II
Subsequently the same procedure was
used to prepare 498 ml of the original stability values for the formulation #2, pH 3.8*
formulation without the addition of
d-pantothenyl alcohol. This was di- Vitamin Percentage retention after
vided into two portions of approx- o day 30 days 60 days 90 days
imately 240 ml each and stored in 12-
Vitamin A 101 .66 91.72 87.43 82.84
fluid-ounce, amber bottles and des- Ascorbic acid 99.80 88.05 79.70 70.41
ignated as formulations five and six. Thiamine hydrochloride 100.05 89.31 84.02 79.02
Formulation five with pH of 3.9 was Riboflavin 100.44 97.93 97.05 95.94
stored in the dark at room temperature. Pyridoxine hydro-
chloride 98.20 96.10 96.30 95.83
Formulation six with a pH of 3.9 Nicotinamide 98.16 97.20 94.10 92.21
was stored in the dark at 37 degrees C Vitamin Bl2 100.13 83 .23 71.41 60.04
with the air above the solution being
replaced by nitrogen. Each time the * Formulation was incubated in the dark at 37 0
in an amber colored bottle .
bottle was opened, nitrogen was again
introduced above the solution . Table III
Since there might be some doubt as
to the safety of polyethylene glycol stability values for the formulation #3, pH 2.8*
200 for internal use, the approval of the
Food and Drug Administration would Vitamin Percentage retention after
be needed before a product as suggested o day 30 days 60 days 90 days
could be placed on the market. How-
ever, single acute oral dosageS to rats is Vitamin A 101 .66 92.17 85.31 79 .01
Ascorbic acid 99.80 89.22 79.44 69.11
of an extremely low toxicity of the order Thiamine hydrochloride 100.05 91.57 85.74 80.41
of 28.9 ml/kg LD 50. Repeated oral Riboflavin 100.44 96.72 96.21 94.27
feedingS in the diet of rats for a period Pyridoxine hydro-
of two years found an acceptable level chloride 98.20 97.30 97.11 94.31
Nicotinamide 98.16 97.17 96.89 96.70
of four percent. Vitamin B l2 100.13 82.27 70.43 59.37
Each sample was assayed for all
vitamins except Vitamin D and * Formulation was incubated in the dark at 37 0
in an amber colored bottle .
d-pantothenyl alcohol immediately after
preparation and then at 30-day inter- formulations were expressed in terms of proportions with water and with sorbitol
vals. All the assays were run in percent retention for each of the solution.
duplicate and results are given in vitamins studied as given in tables I, In the formulations stored at 37
averages. When the duplicate results II, III, IV, V and VI. degrees, the lower the pH value within
varied more than five percent, the assays limits, the greater the stability of
discussion
were repeated until satisfactory results thiamine hydrochloride. Formulations
were obtained. The experimental data of this in- two, three and four, which were similar
The analytical methods used were vestigation indicated that at least except that the respective hydrogen
those of USP XV except for the assay four factors influenced the stability ion concentrations expressed as pH
of Vitamin B12 which was the method of values of the vitamins in the formula- were 3.8, 2.8 and 4.8, gave percent
Rudkin and Taylor 6 and for nicotin- tions. These are (1) storage tempera- retention of thiamine hydrochloride of
amide which was the method of Lamb 7• ture, (2) vehicle, (3) pH of the for- 79, 80 and 69 respectively, when the
The instruments used were a Paltz mulation and (4) oxygen of the air. formulations were incubated at 37
and Bauer Model B Fluorophotometer With respect to the temperature, it degrees for 90 days. The difference in
for the fluorometric assay of thiamine was found that storage at 37 degrees retention of thiamine hydrochloride at
hydrochloride and riboflavin, a Bausch hastens the rate of deterioration. pH 2.8 and 3.8 was very slight in
and Lomb Spectronic 20 colorimeter The vehicle used in this investigation these similar formulations.
for colorimetric assay of nicotinamide was polyethylene glycol 200 and pro- The high loss of thiamine hydro-
and pyridoxine hydrochloride and a pylene glycol mixture (3:1 v/ v) con- chloride in these formulations may be
Beckman Model DU Spectrophotometer taining about three percent of recently attributed to the presence of heavy
for absorption analysis of Vitamin Bu bottled and cooled distilled water. In metals, oxidation and the presence of
and Vitamin A. previous studies 3 •4 propylene glycol decomposition products of ascorbic
The stability values of each of the and glycerin were used in varying acid. One report8 states that heavy

Vol. NS4, No.2. February 1964 87

 Table IV with the B complex even at higher con-
centration although more recent work
stability values for the formulation #4, pH 4.8* may indicate that the decomposition
products of vitamin Bl may cause
Vitamin Percentage retention after degradation of the vitamin. There-
o day 30 days 60 days 90 days fore, the presence of ascorbic acid in
rather high concentration and its
Vitamin A 101.66 89.95 84.97 80.44
Ascorbic acid 99.80 89.23 80.21 72 .57 decomposition products along with
Thiamine hydrochloride 100.05 87.97 78.67 69.34 other factors possibly accounts for the
Riboflavin 100.44 98.11 97 .91 95.11 low retention of vitamin Bl2 in these
Pyridoxine hydro- formulations.
chloride 98.20 97.87 96.59 94.13
Nicotinamide 98.16 96.53 94.07 93 .10 Formulations one and six, which
Vitamin B'2 100 .13 84.71 74 .02 68.41 were similar to formulations five and
two respectively except that the air in
* Formulation was incubated in the dark at 37° in an amber colored bottle. them was replaced by nitrogen before
incubation, retained higher percentages
Table V of all the vitamins. It could be con-
cluded that oxygen of the air hastens
stability values for the formulation #5, pH 3.9* the rate of deterioration.
The stability values for vitamin A
Vitamin Percentage retention after
in all the formulations ranged from
oday 30 days 60 days 79 to 86 even in the presence of anti-
Vitamin A 99.49 93.37 89.11
oxidants in the formulations .
Ascorbic acid 100.05 94.50 91.44 The riboflavin was highly stable in
Thiamine hydrochloride 99.23 92.98 88.91 all the formulations studied. The per-
Riboflavin 101.49 99.89 98 .59 centage retention ranged from 94 to 96
Pyridoxine hydrochloride 101.23 100.74 100.01
Nicotinamide 98.73 98.50 97.21 in the first four formulations which
Vitamin B'2 99.67 86.45 77 .31 were incubated for a period of 90 days.
In all the formulations the stability
* Formulation was incubated in the dark at room temperature in an am ber colored values of nicotinamide and pyridoxine
bottle.
hydrochloride were greater than 92 and
94 respectively.
Table VI
conclusion
stability values for the formulation #6, pH 3.9* A multivitamin formulation using a
Vitamin Percentage retention after vehicle of a mixture of polyethylene
glycol 200 and propylene glycol (3 ; 1
o day 30 days 60 days
v/ v) with only three percent added
Vitamin A 99.49 91.39 87 .51 water containing added antioxidants
Ascorbic acid 100.05 90.81 83 .64 and chelating agents if stored in amber
Thiamine hydrochloride 99.23 89 .94 85.57
Riboflavin 101.49 99.44 98.84 bottles at approximately 25 degrees C
Pyridoxine hydrochloride 101.23 99.87 99.81 at a pH of 3.8 and with the air above
Nicotinamide 98.73 98.61 96 .74 the solution replaced by nitrogen can be
Vitamin B'2 99.67 84.05 74.59 expected to retain its labeled amount of
vitamins for a period of at least three
* Formulation was incubated at 37° in the dark in an amber colored bottle in which the months if excesses of vitamins in the
air was repla ced by nitrogen.
following percentages are used in
preparing the formulation-Vitamin A,
metals are not completely chela ted at under thiamine hydrochloride and prob- 15 percent ; Ascorbic acid, 10 percent;
low pH when the amount of chelating ably also affects ascorbic acid stability. Thiamine hydrochloride, 11 percent ;
agent is as low as 0.01 percent w /v as Bandelin and Tuschhoff9 report Riboflavin, 5 percent; Pyridoxine hy-
used in this investigation. Also, in that in multivitamin liquid prep- drochloride, 4 percent; Nicotinamide,
all of these formulations a large amount arations, the ascorbate ion is more 5 percent and Vitamin B 12 , 30 percent.•
of ascorbic acid decomposition took susceptible to oxidative decomposition.
place, the result probably of an oxidative In this study, the ascorbic acid was references
decomposition which may have had partially converted to sodium ascorbate
1. Stone, A.B ., JAPhA, Sci . Ed., 39, 159
some adverse effect on the stability of which could provide the ascorbate ions (1950).
thiamine hydrochloride. except in the formulation at pH 2.8. 2. Campbell, J .A., ibid., 44, 600(1955).
The percentage retention of ascorbic It was found that the higher the 3. Delgado, J. N., Lofgren, F.V., and Burlage,
H .M., Drug Standards, 26, 5 1(1958).
acid in formulations two, three and four pH value within limits, the greater the 4. Parikh, B.D ., a nd Lofgren, F .V., ibid., 26,
were 70, 69 and 72 respectively. It stability of vitamin B 12 . Formulations 56(1958).
5. "Carbowax, Polyethylene Glycols," Tech-
could be concluded that variations of two, three and four, which were similar ni cal Bulletin, New York, N.Y. , Union
pH between 2.8 to 4.8 have very little except that the respective hydrogen ion Carbide Chemicals Co ., Div. of Union
Carbide Corp., 6 1(1960 ).
effect, if any, on ascorbic acid stability. concentrations expressed as pH were 6. Rudkin, G.O., a nd Taylor, R .J ., Anal .
The following reasons seem to apply 3.8, 2.8 and 4.8, gave percent retention Chem. 24, 1155 (1952).
with reference to ascorbic acid stability of vitamin B12 of 60, 59 and 68 re- 7. Lamb, F.W., ibid., 15, 352(1943) .
8. " The Versenes," Technical Bulletin 12,
-the presence of heavy metals, ox- spectively when the formulations were Framingham, Mass., Bersworth Chemica l
idation and the presence of ascorbate incubated at 37 degrees for 90 days. Co ., 6 1(1951).
9. Bandelin , F.J., a nd Tuschhoff, J .V., JA PhA,
ion except in the formulation at pH Vitamin Bn has been reported 10 as Sci. Ed. , 44, 244(1955).
2.8. The inability of low concentrations incompatible with ascorbic acid. Gam- 10 . "Vitamin Bn, Service Bulletin ," Rahway.
of chelating agent to function effectively bier and Erwin l l report that vitamin N . J ., Merck & Co., Part I , 3(1953).
1l. Gambier, A.S., and Erwin, P .G.R ., JAPhA,
at low pH has already been mentioned Bl2 is perfectly stable in combinations Sci . Ed ., 46,1 40(1957).

88 Journal of the AMERICAN PHARMACEUTICAL ASSOCIATION