Scientific Edition

JOURNAL OF THE
AMERICAN PHARMACEUTICAL
ASSOCIATION
EDITOR,WASHINGTON,
JUSTIN L. POWERS, D. C.

NUMBER 7
VOLUMEXXXIX JULY, 1950
CONSECUTIVE
No. 14

Pharmaceutical Studies with Thiamine

Mononitrate*
By THOMAS J. MACEK, BEATE A. FELLER, and EDWARD J. HANUSt
Thiamine mononitrate, the salt formed from one mole of thiamine base and one
mole of nitric acid, occurs as an anhydrous crystalline solid of low hygroscopicity,
differing in this respect from thiamine hydrochloride. The stability of thiamine
mononitrate was investigated in sterile solutions, compressed tablets, multivitamin
capsules and dry-filled capsules. Assay data are given to show that thiamine mono-
nitrate is more stable than thiamine hydrochloride in these preparations. The use
of thiamine mononitrate in B complex capsules eliminated the need for an acidic
stabilizer, the presence of which accelerated the decomposition of the pantothenic
acid component.

HIAMINE HYDROCHLORIDE U. S. P is com- stable for a considerable time even though pre-
mercially synthesized as thiamine chloride cipitation sometimes occurs. In a number of food
hydrochloride and crystallizes from alcoholic- products and in certain pharmaceutical forms,
aqueous solutions as the hemihydrate (m. p. = particularly in mixtures with other vitamins,
248-250 with decomposition) (1). The molar thiamine hydrochloride, however, has presented
proportion of water of crystallization, however, is stability problems.
not constant during storage and may vary be- A compound which has been found to possess
tween one-half and approximately one, depending some interesting properties as regards stability, is
upon humidity conditions. A moisture content the thiamine salt formed from one mole of thia-
not exceeding 5 per cent of the solid is permitted mine base and one mole of nitric acid. This com-
by the U. S. P. (2). In spite of its variable mois- pound, called thiamine mononitrate, has the
ture, crystalline thiamine hydrochloride as a solid following structure : .
is stable for extended periods a t room tempera-
ture and at elevated temperatures even in contact CH c
’
N
L H 2
/s\
HC C-cH2-cH20H
with air. Aqueous solutions of thiamine hydro- I II II
chloride in the pH range 2.5 to 4.5 likewise are N C-CH2-N-C-CHa
+
* Received January 12 1950 from the Research Labora- \C/
H Nos-
tories, Merck & Co., Inc.: RahAay, N. J.
t We are indebted t o the Microbiology Dept., Research
an$ Development Division, Merck & Co., Inc., for the cal-
cium ntothenate assays, and to the Gelatin Products CO., Thiamine mononitrate is readily crystallized
Inc., Ketroit, for the preparation of the multivltamin cap- from water, leading to the production of a salt of
sules.
365
366 JOURNAL OF THE AMERICAN
PHARMACEUTICAL
ASSOCIATION
high purity which melts at 196-200" (with de- claving at 120" for twenty minutes. The solutions
composition). The concentration of a saturated were assayed before and after autoclaving and again
after various periods of storage a t room temperature
aqueous solution at 25" is 2.7 Gm. per 100 Gm. of up to six months. The p H of the solutions was like-
water; the PH of such a solution is 6.8 t o 7.1. wise determined a t each assay interval. The pH of
Addition of a strong acid, e.g., hydrochloric or the solutions was in a range incompatible with the
nitric, t o solutions of thiamine mononitrate in- stability of thiamine from the start. As a result
significant losses of thiamine occurred during storage
creases solubility b y virtue of the ionic equilibria
as shown in Table I.
involved. For example, b y the addition of suf- In view of this instability and also because higher
ficient hydrochloric acid to maintain a pH of 4, concentrations of thiamine were more desirable, the
concentrations of thiamine mononitrate as high study was repeated with aqueous solutions contain-
as 18.5 Gm. per 100 cc. can be attained at room ing 100 mg. of thiamine mononitrate per cc., ad-
justed to pH 4.0 by the addition of hydrochloric acid.
temperature. T h e addition of a strong acid re- Table I1 shows the solutions which were prepared.
sults in the neutralization of the free amino
group, the (acidic) pK of which is known by direct II.-COMPOSITIONOF SOLUTIONS
TABLE PREPARED
titration t o be 4.8.
Computed on the basis of molecular weights Per Cc. Solution 1 Solution 2
100 mg. Thiamine mono- 100 Gm. 100 Gm.
1.00 mg. of thiamine mononitrate (mol. wt. nitrate
327.37) is equivalent to 1.03 mg. of thiamine 0.26 cc. 1 N Hydrochloric 260 cc. 260 cc.
hydrochloride. Thiamine mononitrate has a acid
5 mg. Chlorobutanol ... 5 Gm.
moisture content of about 0.1 per cent and a low u. S. P.
hygroscopicity, differing in this respect from thi- Distilled water, 1000 cc. 1000 cc.
amine hydrochloride; i t does not deliquesce even t o make
on exposure to 90 per cent relative humidity.
These properties suggested t h e use of thiamine The 1 N hydrochloric acid was added to approxi-
mononitrate as a more stable form of thiamine in mately 600 cc. of distilled water (or distilled water
pharmaceutical preparations. containing the chlorobutanol) and the mixture was
warmed to about 50'. The thiamine mononitrate
EXPERIMENTAL was dissolved with stirring. The cooled solution
was made to volume by the addition of distilled
All assays for thiamine were done by the U. S. P. water.
thiochrome method (3). The results are expressed Solution 1 was filtered and subdivided into 5-cc.
in terms of the U. S. P. thiamine hydrochloride ampuls and flame-sealed. The ampuls were ster-
reference standard. During test periods the calcium ilized by autoclaving at 120' for twenty minutes.
pantothenate was determined microbiologically by Solution 2 was divided in half. The first portion
the L. arabinosus method of Skeggs and Wright (4). (Part A) was filtered and was subdivided into 10-cc.
The results are expressed as pantothenic acid. ampul vials and stoppered with sleeve-type rubber
Samples were stored a t room temperature protected stoppers. The filled vials were sterilized by auto-
from light and in an electric oven a t 40" as indicated. claving at 120' for twenty minutes. The second
The ampuls and vials used for sterile solutions con- portion (Part B) was sterilized by filtration through
formed t o the U. s. P. requirement for type I glass. a micro-porous procelain candle and was aseptically
Considering the reproducibility of the assay method subdivided into 10-cc. ampul vials and stoppered
and the uniformity of distribution of thiamine in the with sterilized sleeve-type rubber stoppers. All
tablet and capsule preparations, variations m assay solutions were assayed initially for thiamine and
values within the limits of +5% were regarded as again after various periods of storage at room tem-
insignificant. perature and a t 40". The stability data are sum-
Sterile Solutions.-The stability of thiamine marized in Table 111. At pH 4.0 thiamine mono-
mononitrate was studied in unadjusted solutions nitrate is stable in aqueous solution. The solutions
containing 10 mg. and 25 mg. thiamine mononitrate were clear and free of precipitate, and had dis-
per cc., respectively, in distilled water. These solu- colored to only a slight extent after storage a t room
tions, filtered through Pyrex sintered glass Biichner- temperature for one year. Samples of Solution 1
type filters, were subdivided into ampul; which were have also been stored at room temperature for as
flame-sealed. The ampuls were sterilized by auto- long as two years without precipitation.

AQUEOUSSOLUTIONS
-TABLESTABILITY OF THIAMINE
MONONITRATE
IN UNADJUSTED

Theoretical Concn.
per Cc.
7

25 Mg.
-Mg.
$H
per Cc. and % Loss-
10 Mg. 9H
-
Before autoclaving 25.5 7.00 10.2 6.75
After autoclaving 23.6 (-6%) 6.15 9.77 (-2%) 6.20
After 1 mo. a t RT 22.9 (-8%) 5.95 9.28 (-7%) 6.00
After 3 mo. at RT 2 2 . 2 (-11%) 5.75 8.75 (-13%) 5.65
After 6 mo. a t RT 12.1 (-52%) ... 6.99 (-30%) ...
 SCIENTIFIC
EDITION 367
TABLE MONONITRATE
III.-STABILITY OF THIAMINE IN A Q ~ O USOLUTIONS
S TO pH 4.0
ADJUSTED
-Mg.per CC. and % Loss
Solution 2
-----Solution -1 , Part A Part-B
Sterilization Autoclaved flH Autoclaved #H Filtered flH
Theoretical concen- 100.0 ... 100.0 ... 100.0 ...
tration
Initial assav
~
~~~~~~~~~ ~ ~ ~ . 99. 5 4-. nn
. in2
- o
-- . - - .gn
3 -- -.- n
102. 3 9fi
After 6 mo: a t R T 96:8(-3%) 4.00 94.5(-7%) 3.84 lOZ.O(O) 3.90
After 12mo. at R T 9 3 . 2 (-670) 3.83 96.6 (-5%) 3.70 97.3 (-5%) 3.71
After 1 mo. at 40" 9 5 . 5 (-4%) 3.94 92.3 (-10%) 3.80 94.7 (-7%) 3.80
After 6 mo. a t 40" 8 9 . 4 (-10%) 3.88 8 6 . 4 (-i5%j 3.66 92.7(-9%j 3.64

Compressed Tablets.-The stability of thiamine intervals. The data summarized in Table V indi-
mononitrate was studied in B complex tablets. cate that thiamine mononitrate was stable in these
The preparations shown in Table IV, with and with- compressed tablets containing other vitamins of the
out ascorbic acid, respectively, illustrate the com- B complex.
position and method of manufacture, and are typical Multivitamin Capsules.-A number of experi-
of several tablet formulations tested. ments performed t o determine the stability of
thiamine hydrochloride in commercially available
TABLE
IV.-TYPICAL B COMPLEX
TABLET multivitamin capsules indicated a general lack of
FORMULAS stability of the thiamine component during ex-
tended periods of storage. Data illustrating this
Per Tablet Tablet
Tablet, 1, 2
ME. Gm. G&.
2 . 2 Thiamine mononitrate 2.2 2.2 TABLE
V.-STABILITY OF THIAMINE
MONONITRATE
Sucrose ... 33.0 IN B COMPLEX
TABLETS
33.0 Ascorbic acid 33.0 ...
1 . 7 Calcium pantothenate 1.7 1.7 --Mg. p e r Tablet and % Loss-
Tablet 1 Tablet 2
2 . 0 Riboflavin 2.0 2.0 Initial assay 2.14 2.12
0 . 5 Pyridoxine hydrochloride 0.5 0.5
10.0 Niacinamide 10.0 10.0 After 6 mo. a t R T 2.11 (-1%) 2.18(+3%)
247.5 Sucrose 247.5 247.5 After 12 mo. a t R,T 2.13 (0) 2.15 (+I%)
After 1 mo. a t 40 2.04 (-59') 2.08 (-2%)
40.0 Cornstarch
13.1 Lubricants
40.0 4 0 . 0
13.1 13.1 After 3 mo. at 40" 2.04 (-52) 2.11 (-1%)
~

After 6 mo. a t 40' 2.17 (+1%) 2.13 (0)

350.0 350.0 350.0

A mixture of the calcium pantothenate, riboflavin,
pyridoxine hydrochloride, niacinamide, sucrose, and
cornstarch, all in fine powder, was granulated with
mucilage of acacia. The granulation was dried a t
45' and was screened 16 mesh. A portion of fines
below 40 mesh was collected. The thiamine mono-
nitrate and the ascorbic acid (or the sucrose added in
place of ascorbic acid) were mixed with the fines,
and then with the remainder of the granulation.
The lubricant, which consisted of a mixture of talc,
stearic acid, and calcium stearate, was sifted 80
mesh and was mixed with the granulation. The
granulation mixture was compressed on a single-
punch machine into standard cup tablets, 12/32 inch
in diameter, each weighing 350 mg.
The tablets were stored in screw-capped amber
bottles a t room temperature and a t 40' for stability
testing. They were assayed for thiamine at the
beginning of storage and were reassayed a t various
are given in Table VI. Thiamine mononitrate was
therefore tested for stability in multivitamin cap-
sules. In order t o directly compare the stability of
thiamine mononitrate with thiamine hydrochloride,
two experimental lots of capsules (capsules 1 and 2)
were prepared for study. These differed only in the
thiamine salt used. A third lot (capsule 3). pre-
pared with thiamine mononitrate in a slightly dif-
ferent excipient, was also studied. The composition
of these capsules is shown in Table VII.
The powdered vitamins were mixed with the
melted excipient which consisted of hydrogenated
vegetable oil, corn oil, and beeswax. The mixture
was milled to a smooth paste and was filled into
colored gelatin capsules on an automatic encapsulat-
ing machine. The filled capsules were hardened and
dried.
The pH of the capsules was measured on an aque-
ous extract which was prepared by digesting 4 opened

TABLE
VI.-STABILITY OF THIAMINE
HYDROCHLORIDE
IN MULTIVITAMIN
CAPSULES
r Mg. per Capsule and % Loss
Sample A Sample B Sample C Sample D
Label claim 1.50 1.50 1.50 1.50
Initial assav 1.41
~ ~~ 1.41 1.48 1.32
- .~~

After Zmo: at R T 1.37 (-3%) i:%(-9%) i:ZS(-i4%)

After 4 mo. at RT 1.15 (- 19%) 1.23 (- 13%) 1.34(-10%) 1 . i 5 . ( 1i 3 m
After 6 mo. a t RT 1.08 (-23%) 1.11 (-21%) 1.17 (-21%) 1.05 (-20%)
After 9 mo. at RT 1 01 (-28%) 1.00 ( -29 %) 1.18 (-21 70) 100(-24%)
After 12 mo. a t R T 0.99 (-30%) 0.87 ( -3870) 1.12(-24%) 0.98 (-26%)
368 JOURNAL OF THE PHARMACEUTICAL
AMERICAN ASSOCIATION
TABLE
VII.-COMPOSITION OF MULTIVITAMIN
CAPSULES
.
-
r Per Capsule ---
1 2 3
Thiamine hydrochloride 1.50 mg.
Thiamine mononitrate 1.50 mg.' 1.50mg.'
Riboflavin 2.00 mg: 2.00 mg. 2.00 mg.
Niacinamide 20.00 mg. 20.00 mg. 20.00 mg.
Pyridoxine hydrochloride 0.10 mg. 0.10 mg. 0.10 mg.
Ascorbic acid 37.50 mg. 37.50 mg. 37.50 mg.
Calcium pantothenate 1.09 mg. 1.09 mg. 1.10 mg.
Vitamin A (from fish liver oil) 5000 U. S. P. units 5000 U. S. P. units 5000 U. S. P. units
Vitamin D (from fish liver oil) 500 U. S. P. units 500 U. S. P. units
Vitamin D (synthetic) .... .... 500 U: S: P. units
Excipients. to make 281 mg. 281 mg. 370 mg.
PH = 3.8 3.9 4.8

capsules with 10 cc. of distilled water at about 65'.
The thiamine potency of each preparation was
determined by assay at the beginning of the test
period and again at regular intervals during storage
a t room temperature and at 40'. The capsules
were stored in screw-capped amber bottles. I n
order t o prevent sticking during storage a t the ele-
vated temperature, the capsules were dusted with
talc before being placed in the oven. The stability
of thiamine in these capsules is indicated by the
assay data summarized in Table VIII.
Dry-Filled Capsules.-In order t o evaluate the
stability of thiamine mononitrate in dry-filled, hard
gelatin capsules, two B complex mixtures were pre-
pared. These contained thiamine hydrochloride
and thiamine mononitrate, respectively, plus other
B vitamins and lactose U. S. P. as a diluent, as
shown in the formulas in Table, IX.

TABLE
VIII.-STABILITY OF THIAMINE AND THIAMINE
HYDROCHLORXDE MONONITRATE
IN MULTXYITAMIN
CAPSULES
-Mg. per Capsule and % Loss -7

------Capsule -1 --Capsule 2- --Capsule 3--

Thiamine Salt = Hydrochloride Mononitrate Mononitrate
Initial assav 1.47 1.49 1.47
After 2 mo: at RT 1.45 (-2%) 1.44 (-3%) i%(-i%)
After 4 mo. at R T 1.21 (- 18%) 1.43 (-4%) 1.43 (-3%)
After 6 mo. at R T 1.10 (-25%) 1.34(-10%) 1.47 (0)
After 9 mo. at R T 1.02 (-31%) 1.33 (-10%") 1.41 (-4%)
After 12mo. at RT, 0.99 i-33ojoj 1.32 (-10%) 1.46 f-lxj
After 6 wks. at 40 1.01 (--3i%j 1.37 (-8%j' 1.48 (+lK)
After 3 mo. at 40' 0.72 (-51%) 1.15(-23%) 1.48 (+1%)
After 6 mo. at 40' 0.43 (-71%) 1.01 (-32%) 1.45(-2%)

TABLE IX.-DRY-FILLED CAPSULEFORMULATIONS The powders were mixed with the lactose. Hard
gelatin capsules were filled with each of the mixtures
Capsule 4, Capsule 5, to contain 324 mg. per capsule.
Mg. Mg. For assay the contents of 20 capsules were emptied
Thiamine hydrochloride 1.000 into*a small mortar and the powder was thoroughly
Thiamine mononitrate i :Ooo mixed by trituration. A weighed sample of the
Riboflavin 01500 0.500
Niacinamide 5.000 5.000 mixture was then taken for analysis. The capsules
Pyridoxine hydrochloride 0.125 0.125 were assayed initially and a t regular intervals during
Calcium pantothenate 0.500 0.500 storage at room temperature and 40". The data
Lactose 316.650 316.650 summarized in Table X indicate that thiamine
~ _ _ _
mononitrate was more stable than thiamine hydro-
Total 324.0 324.0
chloride in these capsules.

TABLEX.-STABILITY OF THIAMINE
HYDROCHLORIDE
AND THIAMINE IN DRY-FILLEDB
MONONITRATE
COMPLEX
CAPSULES
Mg. per Capsule and % Loss
Capsule 4 Capsule 5
(Cg. Thiamine HC1) (Cg. Thiamine Mononitrate)
Initial assay 0.95 0.99
After 2 mo. at RT 0.86 (-9%) 0.99 (0)
After 3 mo. at RT 0.78 (-18%) 0.98(-1%)
After 6 mo. at RT 0.78(-18%) 0.94 (-5%)
After 12 mo. a t RT 0.73 (-23%) 0.97 (-2%)
After 1 mo. at 40' 0.45 (-53%) 0.96(-3%)
After 2 mo. at 40' 0.28 (-70%) 0.98 (- 1%)
After 3 mo. at 40' 0.26 (-73%) 0.98 (-1%)
After 6 mo. a t 40' .... 0.90 ( -9%)
 SCIENTIFIC
EDITION 369
TABLE
XI,-OTHER DRY-FILLED
B COMPLEX
CAPSULE
FORMULATIONS

6 7
Capsule, M g .
a 9
- 10
Thiamine hydrochloride 3.0 3.0 ... ... 3.0
Thiamine mononitrate ... ... 3.0 3.0 ...
Riboflavin 4.0 4.0 4.0 4.0 4.0
Pyridoxine hydrochloride 1.5 1.5 1.5 1.5 1.5
Calcium pantothenate 10.0 10.0 10.0 10.0 10.0
Niacinamide 30.0 ... 30.0 ... 30.0
Niacinamide hydrochloride" ... 40.0 ... 40.0 ...
Tartaric acid 0.65
Cornstarch, to make 324 0 : 324 0 : 324 0 : 324 0 : 324.0
Forty milligrams of niacinamide HCI is equivalent to 30 mg. niacinamide.

XII.-STABILITY OF THIAMINEAND CALCIUM

TABLE IN DRY-FILLED
PANTOTHENATE B COMPLEX
CAPSULES
-Mg. per Capsule and % Loss
6 7 8 9 10
Thiamine
Initial assay 2.54 3.08 3.10 3.15 3.01
Affer1mo.atRT 1.58(-38q/) 3.04(-1%) 3.11(0) 3.08(-2%) 2.84 (-6%)
After3mo.atRT 0.61 -76%) 3.02(-2%) 3.10(0) 3.07 (-3%) 2.80 (-7%)
After 6 mo. at RT 0.44 [-83%) 3.02 (-2%) 3.10 (0) 3.00 (-5%) 2.80 (-7%)
After 1 mo. a t 40' 0.82 (-68%) 3.05 (-1%) 2.95 (-5%) 3.15(0) 2.91 (-3%)
After 3 mo. a t 40' 0.53 (-79%) 2.95 (-5%) 3.02 (-4%) 2.86 (-5%)
After 6 mo. a t 40" 0.44 (-83%) 3.00'(-3%) 3.00 (-3%) 3.00 (-5%) 2.85(-5%)
Pantothenic Acid
Initial assay 10.6 10.3 12.0 10.4 10.5
After 1 mo. a t RT 10.6 (0) 10.0 (-3%) 12.0 (0) 9.1 (-13%) 9.7 (-8%)
After 3 mo. a t R T 10.6 (0) 9.5 (-7%) 12.2 (0) 8.5(-18%) 7.9 (-25%)
After 6 mo. at RT 10.6 (0) 7.3 (-29%) 12.3 (0) 6.8 (-35%) 6.9 (-34%)
After 1 mo. at 40' 10.7 (0) 6.9 (-33%) 11.9 (-1%) 6.0 (-42%) 6.5 (-38%)
After 3 mo. a t 40' 2.4 (-77%) 11.5 (-3%) 2.2 (-79%) 1.7 (-84%)
After 6 mo. a t 40' 10 .*6i0) .... 11.0 (-9%) 1.3 (-88%) 0.3 (-97%)

In order to stabilize thiamine hydrochloride in B SUMMARY

complex mixtures it has sometimes been the prac-
tice to add an acidic substance such as tartaric acid.
Niacinamide hydrochloride has also been used in
this way. Invariably the thiamine hydrochloride is
stabilized but the rate of decomposition of other
vitamin components of the mixture, notably calcium
pantothenate, is increased. With thiamine mono-
nitrate the addition of an acidic stabilizer becomes
unnecessary. This was demonstrated by an experi-
ment in which the thiamine and calcium panto-
thenate stability in a series of five dry-filled B com-
plex capsules was tested during storage at room
temperature and a t 40". The capsule preparations
were made containing variations of thiamine hydro-
chloride and thiamine mononitrate with niacina-
mide, niacinamide hydrochloride, and tartaric acid
as shown in Table XI.
Hard gelatin capsules were filled with each mix-
ture to contain 324 mg. per capsule. The contents
of 20 capsules were mixed and sqmpled for each
assay. The thiamine and pantothenic acid assay
data for each of the capsules are summarized in
Table XII.
Pharmaceutical studies with thiamine mono-
nitrate are reported. The addition of an acidic
stabilizer in B complex capsules was found to be
unnecessary when thiamine mononitrate was used.
It was found to be more stable than thiamine
hydrochloride in the multivitamin capsules and
the dry-filled B complex capsules studied. Thia-
mine mononitrate was found to be stable in com-
pressed tablets and in sterile aqueous solutions
at PH 4.0. Some of the sterile solutions have
been stored at room temperature for as long as
two years without precipitation.
REFERENCES
{I) "The Story of Vitamin Bi," Mzrck & Co., 1940,p.. 15.
2) "United States Pharmacopceia, Thirteenth Retimon,
Mack Printing Co.,Easton. Pa., 1947:. p. 569.
(3) "United States Pharmacopceia Thrteenth Revision,
Mack Printing Co., Easton, Pa.. leaf. p. 705.
4) Skeggs, € R.,
I .and Wright, L. D., J . B i d . Chem., 156,
SllS44h