Stabilization ()f 'Vitamins in Pharmaceutical Preparations

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Stabilization ()f 'Vitamins in Pharmaceutical Preparations:

Part l~l-iifiuence of Common Vehicles on the

~tipility of Vitamins A, B

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M~ C. UPRETY*& V. K. MOHAN RAO With the technical assistance of S. K. Bose Central Drug Research Institute, Lucknow

Manus~r£pt received 2 June 1959

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The stabilities' afforded by different combinations of bases like sugar syrup, propylene glycol, glycerol, sorbitol, etc., to vitamins A, B, and C at 37°C. have been studied. Sorbitol-propylene glycol-glycerol-ethyl alcohol (2: 1 :'1 : 1), syrup-glycerol-water (2: 2: 1) and syrup-glycerol (1: 1) combinations retain 70 per cent of vitamin A, 73 per cent of vitamin B, and 51 per cent of vitamin C respectively during five months of storage, Vitamin C keeps well in the pre

sence of vitamins A and B, in syrup-water (85: 15) and syrup-propylene glycol (1: 1). Syrup-glycerul-water (2: 2: 1) ~ives the best stability to a preparation containing vitamins A, B, and C. In general, sugar syrup affords considerable stability to all the three vitamins in different combinations with other vehicles.

V

I TA MI NS, in multivitamin preparations, are The combinations of the bases used in these studies known to lose their potency during storage", ,are given in Table 1.

. Some of the factors that may be responsible Different fofulUlations prepared contained O~2 per for the instability of vitamins are: (1) the nature of cent benzoic acid as a preservative and the following the base²^{, 3}and the concentration of water present in

the preparation, (2) the

pH

of the mediums, (3) in

compatibility of vitamins's", (4)the presence of certain TABLEt -COMBINATIONS OF BASES EMPLOYED AS VEHICLES FOR VITAMINS metals like Fe, Cu, Ca7,B and stabilizers'r-' like anti

oxidants, metal binders, proteins, amino acids, etc., (The proportions of the vehicles in the combinations arc expressed on uot.iuot. basis)

and (5) conditions of storage like the container'>,

temperature, light, etc. Keeping these factors in COMBINATION ^BASE

view, studies have been undertaken on the stabi No.

lization of vitamins A, B₁and C, when present singly ₁ Syrup 50% + glycerol 50%

and in combination, in different common vehicles 2 Syrup 50% + propylene glycol 50%.

3 Syrup 50% + sorbitol 50%

like sugarcane syrup, sorbitol, glycerol, propylene gly _4- Syrup 50%

+

liquid glucose 50%

col, etc., and the results are reported in the present 5 Syrup 80% + (95%) ethanol 20%

6 Syrup 80%+water 20%

communication. ₇ Glycerol 50%

+

propylene glycol 50%

8 Syrup 40% + propylene glycol 40% + water

Experimental procedure 20%

9 Syrup 40%+glycerol 40%+water 20%

Vitamins-Vitamin A acetate/palmitate (E. Merck), 10 Syrup 40%+propylene glycol 20%+glycerol 20%+(95%) ethanol 20%

vitamin Bl> HCl (B.D.H.) and vitamin C (E. Merck) ₁₁ Sorbitol 40% + propylene glycol 20% + glycerol

were used. 20% + (95%) ethanol 20%

12 Syrup 85% + water 15%

Vehicles-Syrup (c. 67 per cent cane sugar), sor ₁₃ Syrup 50% + malt 50%

bitol ~syrup Judex), glycerol (B.D.H.), propylene 14 G-]yceroI50%+(30%) ethanol 50%

15 Syrup 50%+ (30%) ethanol 50 % glycol (B.D.H.), ethyl alcohol (95 per cent), liquid ₁₆ 15% ethanol

glucose (commercial), honey (Paulson Kashmir honey, 17 Propylene glycol 50%+(30%) ethanol 50%

18 Malt 50% + (30%) ethanol 50%

Delhi) and malt (Vitamins Laboratories of India, ₁₉ Honey 50%+(30%) ethanol 50%

Lucknow) were employed. 20 Sorbitol 50% + (30%) ethanol 50%

21 Liquid glucose 50% + (30%) ethanol 50%

*Unichem Research Fellow.

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J _SCI. INDUSTR. RES., VOL. 18C, NOVEMBER 1959

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nounts of vitamins per ml., when present either

TABLE 2-RETENTION OF VITAMIN A ACETATE

one or in combination: vitamin A, +450 J.U.;

tamin B

1, 1·0-1·2 rng.: and vitamin C, 5-6 mg. ^BASE ^RETENTION^(%)OF VITAMINAFTER STORAGE FOR

COMBI- ---'

ween 80 (5 mI.) was added to 100 ml. of the solution _NATION ₁ ₂ ₃ _4- ₅ mtaining vitamin A. The

pH

of each preparation No. month months months months months 'as adjusted to the values given below and then ₁ _96·7 _60'0 _53-9 _40·0 _32-2 tared at 37°C. for the experimental periods: 2 80·0 81-0 46·1 46-0 46·1

3 100·0 70-2 60·8 61-8 61·3

TITAMIN FINAL VITAMIN FINAL 4 100·0 53:9 42·5 38·0 30'3

pH VALUE pH VALUE 5 73·0 53·8 52·0

6 96·7 68·3 66·3 65·1 65·0

A 7'0 3·5 7 77·0 77-0 46'1

B₁ 4'0 6·0 8 46·1 40·0 25-8 22'3

C 4·0 4·0 9 96·0 95·0 61·8 60-0

A+B1 6'0 10 100-0 85·4 40·0 38-2

11 100·0 100·0 100·0 77·0 70'0

Vitamins A and B₁were estimated by the Carr

Price and thiochrome methods's respectively. 2,4-,

TABLE 3 - RETENTION OF THIAMINE

Dinitrophenyl hydrazine method, as modified by HYDROCHLORIDE

Meyer et at.H , was adopted for the estimation of

BASE RETENTION (%) OF VITAMIN AFTER STORAGE FOR

vitamin C. COMBI- - - - A . . - _,~

NATION 1 2 3 4 5

Results ^No. ^month ^months ^months ^months ^months

In order to study the stability of vitamins A, B1 1 100·0 98·3 61·0 ·59·0 58·0

2 100'0 100·0 70·0 68·8 46·0

and C in seven different bases in the presence of 15 ₃ _100·0 _100·0 _38·7 _41'8 _31·2 per cent alcohol (Table 1, combinations 14-21), the 5 91·7 . 78·3 41·7 38'8 33·3

6 100-0 78·2 58·1 54-8 54-0

vitamin concentrations were separately estimated at ₇ _100·0 _..J6·4 _25·1

different intervals. 8 100·0 100·0 98·0 95·6 65'2

9 100·0 100·0 100-0 100'0 73·0

The following conclusions can be drawn from these ₁₀ _69·0 _60·0 _55·0 _40·0 _32'1

results: 13 86·0 75·0 43·0 40·0 40·0

(1) Vitamin A is completely lost after 12 weeks in

all the bases except in 15 per cent alcohol, 50 per cent TABLE.4-RETENTION OF VITAMIN C

syrup plus 15 per cent alcohol and 50 per cent pro

BASE RETENT~N (%) OF VITAAllN AFTER STORAGE FOR _-,

pylene glycol plus 15 per cent alcohol, in which the COMBr- (. "--

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NATION 1 2 3 4 5

percentage retentions of the vitamin were 42, 24 and

No. month. months months months months

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24 respectively,

(2) Vitamin B₁deteriorates up to 90 per cent in all ¹ ^95·0 ^66·0 ^62-3 ^60·0 ^51·0 2 93·0 100·0 100'0 76·9 4-:: D

the bases tried after two months. ₃ _78·0 _69·0 _64·1 _40·0 _35·0

(3) Glycerol plus alcohol gives the best protection ⁵ ^92·0 ^78·0 ^67·3 ^52·8 ^19·0

6 69·0 61·0 57-8 24·7 12·0

, to vitamin C during a .storage period of nine weeks. ₇ _92·0 _65-0 _62·5 _60-8 _42·0 Syrup, malt and propylene glycol in the presence of ⁸ ^70·0 ^51·0 ^39'8 ^43·2 ^24-·0

9 72·0 45·0 43-2 37·0 32·0

alcohol, however, were next in order. ₁₀ _56·0 _54·0 _53·0 _53-5 _28·0 (4) Honey is not a suitable base.

Since syrup is cheap and at the same time provides

generally a good protection compared with other gives better protection to vitamin A than glycerol.

bases, it was given preference in later studies. But this finding did not hold good for combinations The stability of vitamin A in 11 formulations was. 8 and 9 where more water was present. It is of examined by determining the vitamin A concentra interest to note that when syr:up was substituted by

tion at different storage periods up to five months. sorbitol, combinations 10 and 11, the stability of _-~,^\1 The results given in Table 2 show that base combi vitamin A increased.

nation 11 gave the best protection, viz. 70 per cent The stability of vitamin B₁in different vehicles was retention of vitamin A. The next best in the order studied for a period of five months. The results of percentage retention were combinations 6, 3 and 9. recorded in Table 3 show that:

Between syrup-water and syrup-alcohol combinations, (1) Combination 9 is the best with a vitamin reten

. the former was found to be better. Further, combi tion value of 73 per cent. Next in order: were combi

nation 2 was a better vehicle than 1 and thus it in nations 8, 10, 6, 2, 4, etc.

directly confirmed the finding drawn from the preli (2) A comparison of the pairs of combinations 9

minary experiments, namely that propylene glycol and 8 and 1 and 2 showed that glycerol with syrup _{" I ,}

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UPRETY & MOHAN RAO: VITAMINS IN PHARMACEUTICAL PREPARATIONS gave better stability to vitamin B_l than propylene

glycol under similar conditions.

(3) In the presence of syrup, water provided better stability to vitamin B_l than alcohol (combinations 5 and 6).

In Table 4- are presented the percentage retentions of ascorbic acid in some vehicles at various intervals of storage. The results indicate that: (1) the highest percentage retention of vitamin C was in combination 1, viz. 51 per cent, followed by combinations 2, 7, 3, 9; (2) in the presence of syrup, alcohol provided better stability to vitamin C than water (combina

tions 5 and 6) and (3) of the two combinations 1 and 2, the former provided greater stability to vitamin C.

The stability afforded by three different vehicles to vitamins B₁ and C when present together was examined and the percentage retentions of vitamins at different storage intervals are recorded in Table 5.

The results show that combination 1 was the best with retention values ot 38 per cent tor vitamin Bl

and 74 per cent for vitamin C after 15 weeks of storage. The retention values for vitamins B_l and C after three months' storage showed combination 1 to be the best. It was also noted that in all the three combinations tried, vitamin C kept well in the presence of vitamin B_{l •}

The best combination of vehicles fora preparation containing both vitamins A and C seems to be combi

nation 12 with retention values of 58 per cent for vitamin A and 84 per cent for vitamin C (Table 6).

In combination 1 also vitamin C was fairly stable in the presence of vitamin A.

The stability of vitamins A and B₁when present together in three different vehicles was examined and the results are tabulated in Table 7. The results show that: (1) vitamin A deteriorates in all the com

binations in the presence of vitamin B_{l ,}which, how

ever, kept well and (2) the best combination of vehicles was 2 with respect to the stability of vitamin B₁and combination 12 with respect to vitamin A.

It was observed that all the three vitamins A, B_l , /

and C were reasonably stable in all the vehicles after storing them for 13 weeks (Fig. I), even though vitamin B₁ was relatively less stable under these conditions. The best combination was 9 consisting of syrup-glycerol-water (2: 2: 1), in which the per

centage retention of vitamins A, B_l and C was 70, 51·6 and 78·5 per cent respectively.

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Discussion

On examining the effect of shelf-life on the potency of vitamins in multivitamin preparations, Campbell-"

observed that vitamins A, B_{1 ,} B₁₂and pantothenic acid were markedly affected by storage. In a recent communication Delgado et al.³reported that vitamins A, B_l and C in a multivitamin preparation deteriorat

ed by about 50, 50 and 35 per cent respectively in a vehicle consisting of propylene glycol-glycerol-water (2: 2: 1), even though the preparation contained a metal binder (a salt of ethylenediamine tetracetate).

TABLE 5-RETENl'ION OF VITAMINS B₁AND C WHEN PRESENT TOGETHER

BASE _{, - -}RETENTION (%) OF VITAMIN B 1 AFTER STORAGE FOR ~ A . . _{____} RETENTION (%) OF VITAMIN C AFTER STORAGE FOR

COMBINA \ r - - - - A... ~

TION-No. 4 weeks 8 weeks 12 weeks 15 weeks 4 weeks 8 weeks 12 weeks 15 weeks

1 100·0 78·3 44·6 100·0 106·6 102·0

2 81·0 45·0 45·0 38-0 100·0 93'3 82·5 74·0

12 67·0 67·0 47·6 19·0 106·0 75'3 76·0 76·0

TABLE 6-RETENTION OF VITAMINS A (ACETATE) AND C WHEN PRESENT TOGETHER

BASE RETENTION (%) OF VITAMIN A AFTER STORAGE FOR RETENTION (%) OF VITAMIN C AFTER STORAGE FOR

COMBINA- r- .... ---, ,---- "- ---..

TION No. 4 weeks 8 weeks 12 weeks 15 weeks 4 weeks 8 weeks 12 weeks lS weeks

2 87·0 78·0 33-1 29·0 38'4 38·0 38·0 35·0

1 84·0 87·0 60'4 37·0 100·0 100·0 100·0 95·0

12 84·0 75·4 58'01- 58·0 100·0 95·0 84·2 84·0

TABLE 7-RETENTION OF VITAMINS A (ACETATE) AND B ₁WHEN PRESENT TOGETHER

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BASE RETENTION ('Yo) OF VITAMIN A AFTER STORAGE FOR RETENTION (%) OF VITAMIN B1 AFTER STORAGE FOR

COMBINA- "- r - - - A. ,

TION No. 4 weeks 6 weeks 12 weeks 15 weeks 4- weeks 6 weeks 12 weeks 15 weeks

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1 62'1 30·5 10'4 6'5 71-4 70·0 62·8 57·1 (

2 60-0 29'2 8·5 5·9 90·0 83·4 82·0 83·4

12 58·0 58·0 28·0 24·0 52·0 50·0 44'6 42·3

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COMBINATION .2 COMBINATION 7 COMBINATION ~ STORAGE PE:RIOO: ~ AT START, ~ 4WtEKS.~ 9 WEEKS,U'l.3WEEKS.

FIG. 1 - RETENTlON OF VITAMINS A (PALMITATE), B 1 AND C WHEN PRESENT TOGETHER IN DIFFERENT COMBINATIONS OF

BASES DURING STORAGE

Considering the results obtained in the present study in the light of the above reports, it may be seen that vitamins A and C deteriorate to the extent of 20-25 per cent (except in one combination wherein the deterioration of vitamin C was about 45 per cent) as shown in Fig. 1. Also, the experimental conditions employed in our studies differ from those of Delgado et al.^3• Even though thiamine deteriorates marked

ly relative to vitamins A and C, a retention value of 51 per cent was obtained for vitamin BI in the combination 9.

" The high stability values obtained for vitamin A (Fig. 1) may be due to the fact that vitamin A pal

mitate instead of acetate was used in these experi

ments. This finding is well supported by a recent report of Ponci and Gialdi'" who stated that palmitic ester of vitamin A is more stable than the acetic ester in aqueous preparations.

Vitamin C is quite stable in the presence of vita

min B_l (Table 5) while the latter undergoes pro

nounced degradation. This corroborates the findings of Dutta et

at.

¹⁵ and Ito et

at.

5 • From the results given in Table 5 it can be seen that the concentrations of vitamins A and C are more or less maintained in different combinations of vehicles. Hence, it appears that vitamins A and C do not individually contribute towards their respective stabilities.

Among the various vehicles investigated (Tables 1 and 4), sucrose, glycerol and propylene glycol have stabilizing effect on ascorbic acid. This conclusion agrees with the findings of Bandelin and Tuschhoff'".

It is clear from a study of pairs of combinations 1, 9 and 2, 8 (Table 4) that the stability of ascorbic

VOL. 18C, NOVEMBER 1959

acid is influenced by the amount of water present in the combinations. Gerber and co-workersl'' reported a similar finding and stated that the decomposition of vitamin C in the presence of vitamin BIz- and ferrous gluconate could be prevented by using 70 per cent sorbitol as a base. In order to explain this.

they postulated that sorbitol formed complexes with vitamins making them less susceptible to degradation or bound water so strongly that it was less available for chemical interaction. In this connection further work has already been started with a view to explain the mechanism by which vitamin C deteriorates in the presence of water.

Unlike vitamin C, thiamine is more stable in the presence of water (Table 3). This inference, though contradictory to that of Delgado et al.3, confirms the observation of Stone",

It was found that vitamin A acetate deteriorates appreciably in the presence of thiamine (Table 7).

But this is not in agreement with the finding of Katsuiw, who reported that vitamins BI, BIz, C and D had no effect on the stability of vitamin A. The deterioration of vitamin A acetate under the experi

mental conditions employed in this study may be due to the

pH

of the preparations. Experiments are now in progress to verify this.

It may be concluded, from the above that cane sugar syrup as a base affords good stability

10

^vita

mins A, B₁and C.

Summary

(1) Good stabilities were provided by vehicles sorbitol-propylene glycol-glycerol-alcohol (2: 1: 1: 1), syrup-glycerol-water (2: 2: 1) and syrup-glycerol (1 :1) to vitamins A, B_l and C respectively during storage for five months at 37°.

(2) Vitamin B₁deteriorated markedly within four months in the presence of vitamin C, but the latter was fairly stable in the formulation syrup-propylene glycol (1: 1).

(3) Syrup-water (85: 15) as a vehicle afforded fair stability of vitamin C in the presence of vitamin A.

(4) Vitamin A deteriorated appreciably in the pre

sence of vitamin B_l

(5) Syrup-glycerol-water (2: 2: 1) as the vehicle gave the best stability to a preparation containing vitamins A,

s,

and C.

(6) Syrup as a vehicle afforded considerable stabi

lity to all the three vitamins in different combinations.

References

1. MOHAN RAo, V. K., Stabilization of Vitamins and Its Importance to Pharmaceutical Industry, paper presented at the Symposium on Scientific Conservation of Mate

rials, Technical Development Establishment, Kanpur,

1959.

2. STONE, G. B••J. A mel'. pharm. Ass. (Sci. Ed.), 39 (1950),159.

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LETTERS TO THE EDITOR 3; DELGADO, j. N., LOFGREN, F. V. & BURLAGE, H. M.,

Drug Siand., 26 (1958), 51.

4. MACEK, T. j., FELLER, B. A. & HANUS, E. ] .• ]. Amer.

pharm, Ass. (Sci. Ed.), 39 (1950), 365. ⁰ 5. ITo, A., INAJIH, K. & OHARA. A., Chem, Abstr., 49 (1955),

16346.

6. PARIKH, B. D. & LOFGREN, F. A., DrugStand.,26 (1958), 55.

7. TAUB, A., KATZ, I. & KATZ, M., ] . Amer, pharm. Ass.

(Sci. Ed.), 38 (1949), 119.

8. DUTTA, N. K., MEHTA, C. j, & NARAYANAN, K. G. A., Indian j. Pharm., 14 (1952), 53.

9. TANSEY, R. P. & SCHNELLER, G. H., J. Amer. phon», Ass. (Sci. Ed.), 44 (1955), 34.

10. NASH, R. A., Amer.]. Pharm., 130 (1958), 152.

11. BRUMFIELD, P. E. & GROSS, H. M.; Drug & Cosmet, Ind., 77 (1955), 46.

Letters to

PARTIAL FAILURE OF MEIOTIC SPINDLE IN SUAEDA FRUTICOSA FORSK.

Suaeda fruticosa FORSK. IS A HIGHLY POLYMORPHIC

species usually inhabiting saline soils. The natural occurrence of diploid, tetraploid and hexaploid races in the species has been recently reported', A morpho

logical variant with bushy habit and flattened leaves was observed in a naturally growing population of the species. The variant was analysed cytologically and found to 'be at the diploid level with meiotic abnor

malities, the significance of which is reported in the present note.

The material for meiotic studies was fixed for 24 hr in Carney's fixative (6: 3 : 1) and then squashed in iron acetocarmine.

At early diakinesis, 9 bivalents were observed.

The number of chiasmata varied from 1 to 2 per bivalent. Bivalents displayed excessive contraction assuming more condensed configurations at meta

phase 1. Pollen mother cells with normal and ab

normal meiosis were seen to occur side by side at this stage. Seven bivalents and four univalents were observed in some P.M.C.'s (PI. 1, Fig. 1). Bivalents did not organize themselves properly at the equa

torial plate and were seen to lie scattered singly or in groups. Anaphase I was abnormal with 18 chromo

somes which were distributed irregularly (PI. I, Figs.

2 and 3). In Table 1 is recorded the variation in the distribution of chromosomes at anaphase 1.

Anaphase II was also irregular. Thirty-six half univalents were noticed in one pollen mother cell.

These were distributed in one to five groups, the dis

tribution in 1-2 groups being more common (PI. I, Figs. 4 and 5). The details of chromosomal distri

bution at anaphase II are given in Table 2.

12. SUBRAHMANYAM, S. V. & MAJESKE, j. P., Amer. J.

Pharm., U9 (1957), 222.

13. The Association of Vitamin Chemists, Methods oj Vitamin Assay (Interscience Publishers Inc., New York), 1951.

14. MEYER, K. H., HASELBACH, C. H. & BOISSONNAS, R. A., Helv. chim. acta, 35 (1952), 1781.

15. DUTIA, N. K., MEHTA, C. j, & NARAYANAN, K. G. A., Indian J. Pharm., 15 (1953), 164.

16. CAMPBELL, ]. A. & McLEOD, H. A., ]. Amcr. pharm, Ass. (Sci. Ed.), 44 (1955), 263.

17. PONCI, R. & GIALDI, F., Chcm, Abstr., 50 (1956), 4458.

18. BANDELIN, F. j, & TUSCHHOFF, j, V., j. Amer. pharm, Ass. (Sci. Ed.), 44 (1955), 241.

19. GERBER, C. F., HETZEL, C. P., KLIOZE, O. & DEYDEN,

A. F.,]. Amer, pharm: Ass. (Sci. Ed.), 46 (1957), 635.

20. RAISUI, G., Cbem, Abstr., 47 (1953), 10174.

the Editor

2

3 4

5

PLATE I - MEIOTIC CHANGES IN S. fruticosa FORSK. X 1420 [Fig. 1; metaphase I; Figs. 2 and 3: anaphase I; and Figs.

4 and 5: anaphase II]

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