GAMMA RADIOLYTIC DEGRADATION OF VITAMIN B1 AND …

www.wjpps.com Vol 6, Issue 6, 2017.

929

GAMMA RADIOLYTIC DEGRADATION OF VITAMIN B1 AND

VITAMIN C

Sailee A. Bhange*1, Dilip V. Parwate

2 and Kiran M. Khandarkar

3

1Yeshwantrao Chavan College of Engineering, Nagpur, Maharashtra, India.

2Department of Chemistry, RTM Nagpur University, Nagpur Maharashtra, India.

3G. H. Raisoni Academy of Engineering and Technology, Nagpur, Maharashtra, India.

ABSTRACT

Radiation chemistry has many novel applications. Earlier it was

considered to have only degradation effect on the materials on which

radiations were incident but the scenario has changed a lot now and

several constructive applications are deliberated using high energy

radiations. Gamma radiation sterilization method is a very clean

procedure as it does not leave behind any chemical products. The heat

labile compounds can be sterilized using these methods. This method

of sterilization can be directly used after final packaging of products

reducing the requirement of strict aseptic conditions at all stages of

production. In the present work we have focused on the radiation induced degradation of

Vitamin B1 and Vitamin C. Thiamine hydrochloride and two vitamin B1 tablets, Beplex Forte

and Neurobion Forte, were used. Ascorbic acid and two vitamin C tablets namely, Celin and

Limcee, were used for the present study. The standards and tablets were irradiated in Gamma

chamber in solid state as well as aqueous phase at the dose rate of ~0.3 kGy/hr and

characterized by IR and NMR. Thiamine hydrochloride was irradiated in solid phase and its

assay was checked spectrophotometrically. The assay of ascorbic acid was studied by

iodometric titration. It was found from FTIR and NMR spectra that irradiation does not cause

any change in the chemical structure. The radiation sterilization of these vitamins can be

effectively carried out in solid state as decomposition of vitamins does not occur due to

gamma irradiations in the solid state. However, aqueous solution of thiamine hydrochloride

gets extensively degraded due to gamma irradiation. Aqueous solution of ascorbic acid is

quite resistant to gamma radiation and does not undergo much radiation damage at low

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 6, 929-952 Research Article ISSN 2278 – 4357

Article Received on

24 March 2017,

Revised on 13 April 2017,

Accepted on 03 May 2017

DOI: 10.20959/wjpps20176-9240

*Corresponding Author’

Dr. Sailee A. Bhange

Yeshwantrao Chavan College

of Engineering, Nagpur,

Maharashtra, India.

http://www.wjpps.com/

Bhange et al. World Journal of Pharmacy and Pharmaceutical Sciences


930

dosages of gamma irradiation. Thus, vitamin C syrups can also be sterilized by low dose of

gamma radiation.

KEYWORDS: Gamma radiation sterilization, Vitamin B1, Vitamin C.

INTRODUCTION

Sterilization process is used for the benefit of human beings which includes the sterilization

of medical supplies and pharmaceuticals. Gamma irradiation sterilization methods stand out,

with numerous advantages. In many countries, it has been accepted as a method of

sterilization and has become routine since it was introduced. With regards to pharmaceutical

products, gamma irradiation sterilization has been used as the better method and records

show the continuous development of the method for sterilization. Recent studies have

focused on many different pharmaceutical products, active and auxiliary substances, active

ingredients in drugs, new drug delivery systems and discuss the effects of radiation

sterilization on the work.

Gamma irradiation is the most important method for the sterilization of pharmaceuticals, due

to the high ability to penetrate the sterile packaging of pharmaceutical and cosmetic products.

The fact that the final heat during the process did not increase their traceability or ability to

deliver effectively is helpful for the heat-sensitive substances in packaging materials or

operations. Gamma rays are also easier to control, secure, reliable and provide a fast process.

The process does not require post-quarantine measures and the product obtained has no

harmful effect on the environment.[1]

Gamma-radiation is increasingly coming into use in place of conventional agents such as heat

and ethylene oxide for sterilization of medical products including pharmaceutical

preparations. However, before this technique becomes acceptable, it is absolutely essential to

establish that radiation does not introduce toxic substances by transformation of either the

active material or the medium in which it is present during radiation sterilization. Many

pharmaceutical preparations e.g. vitamins and antibiotics are often administered in dilute

aqueous media, such as syrups and suspensions. In such systems, the ionising radiation

interacts almost exclusively with water to give hydrated electrons, hydrogen atoms and

hydroxyl radicals and also molecular hydrogen and hydrogen peroxide. Of these, the first

three are very reactive towards many functional groups present in organic molecules that

constitute the active component of the pharmaceutical. Hydrogen peroxide, being capable of




931

acting both as an oxidising and reducing agent, can cause damage to the active component.

Even in the solid state a compound can undergo radiation damage as a result of ionisation and

excitation events resulting from exposure to the ionizing radiation. Therefore, during

radiation sterilization, pharmaceutical preparations may not only lose their potency wholly or

partly but also new substances would be produced whose effect on the organism could be

entirely different from that of the parent. It is therefore very essential to first assess the extent

of radiation damage to the active component and identify the products formed. Secondly,

knowing the reaction pathways that lead to the damage, it should be possible, by the addition

of suitable chemicals or otherwise, to find out ways and means to minimise such radiolytic

transformations.[2]

Gamma radiation is included in the sterilization process (BP 1963 (British Pharmacopoeia)

and USP XVII (U.S. Pharmacopoeia)) and the proposed dose of sterilization is 25 kGy.

However, the level of sterility in some products requires doses of 115 kGy due to the

microbial load (bioburden).[3]

Radiolysis of water soluble vitamins

A vitamin is an organic compound and a vital nutrient that an organism requires in limited

amounts. An organic chemical compound (or related set of compounds) is called a vitamin

when the organism cannot synthesize the compound in sufficient quantities and it must be

obtained through diet. Thirteen vitamins are universally recognized at present. Vitamins are

classified by their biological and chemical activity and not by their structure. Thus, each

vitamin refers to a number of vitamer compounds that all show the biological activity

associated with a particular vitamin. The vitamins are also grouped according to their

solubility as fat soluble vitamins and water soluble vitamins. The fat soluble vitamins are

vitamin A, D, E and K while the water soluble vitamins are vitamins of B complex group and

vitamin C.

Vitamin B includes different vitamins such as Vitamin B1 (Thiamine), Vitamin B2

(Riboflavin), Vitamin B3 (Niacine), Vitamin B5 (Pantothenic acid), Vitamin B6 (Pyridoxine),

Vitamin B9 (Folic acid) and Vitamin B12 (Cyanacobalamin). In the present work we have

focused on the radiolysis of water soluble vitamins.

Thiamine is an essential nutrient required by all tissues, including the brain. The human body

itself cannot produce thiamine but must ingest it with the diet. Thiamine derivatives and




932

thiamine-dependent enzymes are present in all cells of the body. Thus, a thiamine deficiency

would seem to adversely affect all of the organ systems. However, the nervous system is

particularly sensitive to thiamine deficiency, because of its dependence on oxidative

metabolism. Well-known syndromes caused by thiamine deficiency include beriberi,

Wernicke-Korsakoff syndrome and optic neuropathy.

Colovos and Churchill[4]

observed thiamine, riboflavin, pyridoxine, nicotinamide and two

multivitamin preparations containing Ca-pantothenate to be strong enough to be sterilised by

electron irradiation. Rhee[5]

et al. studied effects of gamma irradiation on an argon-saturated

solution of thiamine and found that radiolysis products increased with increasing thiamine

concentration. Kishore[6]

et al. studied the radiation damage and protection of some water

soluble vitamins in aqueous media. Kishore[7-9]

et al. also studied the extent of radiolytic

decomposition of the B-group vitamins, thiamine, riboflavin, pyridoxin, nicotinamide,

pantothenic acid and folic acid in aqueous media as a function of γdose both in the absence

and presence of glucose as a protective additive. Thiamine hydrochloride, riboflavin,

calcium pantothenate, nicotinamide, pyridoxal HCl and cyanocobalamin decay up to 10 kGy

was observed in a study by Jeszka.[10]

Ascorbic acid (vitamin C) is a water-soluble micronutrient required for multiple biological

functions. Ascorbic acid is a cofactor for several enzymes participating in the post-

translational hydroxylation of collagen, in the biosynthesis of carnitine, in the conversion of

the neurotransmitter dopamine to norepinephrine, in peptide amidation and in tyrosine

metabolism. In addition, vitamin C is an important regulator of iron uptake. It reduces ferric

to ferrous ions, thus promoting dietary non-haem iron absorption from the gastrointestinal

tract and stabilizes iron-binding proteins. Most animals are able to synthesise vitamin C from

glucose, but humans, other primates, guinea pigs and fruit bats lack the last enzyme involved

in the synthesis of vitamin C (gulonolactone oxidase) and so require the presence of the

vitamin in their diet. Thus, the prolonged deprivation of vitamin C generates defects in the

post-translational modification of collagen that causes scurvy and eventually death. In

addition to its antiscorbutic action, vitamin C is a potent reducing agent and scavenger of free

radicals in biological systems.[11]

B. S. N. Rao[12]

carried out a study of the effect of γ-radiation on pure ascorbic acid solution

and on its solution containing dissolved solutes and gases. L’ova and colleagues[13]

found




933

that ascorbic acid in the solid form is resistant to 25 kGy of radiation. They reported that the

deterioration and decay increased significantly with increasing concentrations of beta and x-

ray radiolysis of dilute solutions and also that degradation is more common in a nitrogen

atmosphere.

In the present work we have carried out the radiolysis of Thiamine (Vitamin B1) and ascorbic

acid (Vitamin C) in the solid state as well as aqueous solutions. Two tablets of Vitamin B1

and Vitamin C were also irradiated and their assay was checked. Also, their IR and NMR

spectra were recorded.

Water soluble vitamins- Characteristics

Thiamine

Thiamine, thiamin or vitamin B1 named as the "thio-vitamine" is a water soluble vitamin of

the B complex. Its IUPAC name is 3-((4-Amino- 2- methyl- 5- pyrimidinyl) methyl)- 5- (2-

hydroxyethyl)- 4- methylthiazolium chloride with molecular formula C12H17N4OS+[Cl

] and

molecular weight 265.35. The structural formula of the compound is shown in Fig. 1.

Thiamine is a colourless organosulphur compound which is soluble in water, methanol and

glycerol and practically insoluble in less polar organic solvents.

Fig 1: Thiamine hydrochloride.

Ascorbic acid

Ascorbic acid is a naturally occurring organic compound with antioxidant properties. It is

one form of vitamin C. Its IUPAC name is (5R)-[(1S)-1,2-Dihydroxyethyl]-3,4-

dihydroxyfuran-2(5H)-one. Its molecular formula is C6H8O6 and molecular weight 176.12.

The structural formula of ascorbic acid is shown in Fig. 2. It is a white solid, but impure

samples can appear yellowish. It dissolves well in water to give mild acidic solutions.




934

Fig 2: Ascorbic acid.

MATERIALS AND METHODS

Thiamine hydrochloride obtained from Loba Chemie was used for the present study. Two

vitamin B1 tablets were also used namely Beplex Forte and Neurobion Forte obtained from

Merck. Ascorbic acid obtained from Merck and two vitamin C tablets namely, Celin (Glaxo

Smithkline) and Limcee (Abbott Healthcare), were used for the present study.

Thiamine hydrochloride, Beplex Forte, Neurobion Forte, ascorbic acid, Celin and Limcee

were irradiated in Gamma chamber (GC-900, housed in the Department of Chemistry, RTM

Nagpur University) in solid state (as well as aqueous phase) at the dose rate of ~0.3 kGy/hr

and characterized by IR and NMR. FTIR analysis was carried out on Perkin Elmer-Spectrum

RX-IFTIR spectrophotometer at Central Instrumentation Laboratory, Chandigarh. 1H NMR

spectra operating at the frequency of 100 MHz was recorded on a Cryo-magnet (Bruker

Avance II 400 NMR Spectrometer) instrument using tetramethylsilane (TMS) as an internal

standard ( = 0 ppm) with D2O as a solvent at SAIF, Chandigarh. Chemical shifts are

reported in parts per million (ppm scale).

Assay of unirradiated and irradiated thiamine

Thiamine hydrochloride was irradiated in solid phase and its assay was checked

spectrophotometrically. 0.1 mM solution was prepared from irradiated solid thiamine

hydrochloride for recording absorption spectra. Also, for thiamine hydrochloride solution, 0.1

mM solution (unirradiated) was prepared for gamma irradiation. The solvent required for

spectrophotometric analysis of thiamine is phosphate buffer having pH 6.8. The solvent was

prepared by mixing equal volumes of 0.01 M solutions of potassium dihydrogen

orthophosphate and disodium hydrogen orthophosphate. The amount of vitamin decomposed

after irradiation was estimated by recording absorption spectra on Elico SL 210 double beam

UV-visible spectrophotometer in the UV region.




935

The tablets that were used for the study contained B complex vitamins i.e. thiamine,

riboflavin, nicotinic acid, niacinamide, pyridoxine hydrochloride, calcium pantothenate, folic

acid, cyanacobalamin and other substances. All the vitamins of B complex group have

absorption maxima in the range of 200-300 nm. The absorption spectrum of thiamine was

quite overlapped due to presence of other components. It was not possible to extract only

thiamine from the tablets and hence the absorption spectra for the tablets could not be

studied.

Assay of unirradiated and irradiated ascorbic acid

The assay of ascorbic acid was studied by iodometric titration. In the determination of

ascorbic acid, the KIO3 method employs a back titration of standard thiosulphate solution

with I2 generated by adding a known amount of KIO3 solution to ascorbic acid solution.

In this titration ascorbic acid is titrated with standard potassium iodate (KIO3) in the presence

of excess potassium iodide in acidic medium. The iodate ion oxidizes iodide ion to I2 and is

itself reduced to I2 according to the reaction,

IO3 + 5 I

+ 6 H

+ 3 I2 + 3 H2O

The ascorbic acid is oxidized by the liberated iodine to dehydroascorbic acid as follows-

The excess of I2 generated in the KIO3 reaction is titrated against sodium thiosulphate using

starch-iodine complex as indicator.

0.01 M ascorbic acid solution was used for checking the assay while four tablets were

dissolved in 1 L doubly distilled water and used for subsequent radiolysis and assay

determination. 15 mL of the prepared solutions were kept for irradiation in gamma chamber

and amount of ascorbic acid remaining was determined after irradiation.




936

RESULTS AND DISCUSSION

Characterization of standards and tablets by FTIR and NMR

The FTIR spectra of unirradiated and irradiated thiamine hydrochloride, Beplex Forte,

Neurobion Forte, ascorbic acid, Celin and Limcee are shown in Fig. 3 (a-l).

In the IR spectrum of unirradiated and irradiated Thiamine hydrochloride[14]

(Fig. 3 (a and

b)), the broad peak at 3209.11 cm1

represents the primary hydroxyl OH stretching and the

band at 3497.18 cm1

corresponds to the primary aromatic amine group. Methyl asymmetric

stretching is observed at 2966.10 cm1

and 2912.10 cm1

frequency is assigned to methylene

asymmetric stretching frequency. The aromatic tertiary amine CN stretching is observed at

1354.17 cm1

.

The FTIR spectra of ascorbic acid, unirradiated and irradiated, are shown in Fig. 3 (g and h).

The spectra show OH stretching between 3526 and 3031 cm1

and CH stretching at 2916

cm1

whereas OH stretching is observed at 2744 cm1

. C=O stretching is observed at 1754

cm1

while C=C stretching can be seen at 1673 cm1

. CH2 scissor frequency, CH2 wagging

frequency and C-H deformation can be observed at 1456, 1321 and 1275 cm1

respectively.

The peaks at 1221 and 1198 cm1

are observed due to skeletal vibrations. The peaks in the

range of 1076 to 1044 cm1

are assigned to COC stretch while peaks at 1026 and 998 cm1

are possibly due to CC stretch.[15]

The IR spectra for unirradiated and irradiated standards and tablets are almost identical

indicating that gamma irradiation does not cause any chemical change in the vitamins.

RC SAIF PU, Chandigarh

Sailee A B-9.sp - 5/19/2015 - TSU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

0.0

5

10

15

20

25

30

35

40

45

50

54.8

cm-1

%T

3828,53

3497,18

3428,23

3209,11

3052,92966,10

2912,10

2831,122769,13

2079,402013,41

1731,45

1661,71613,9

1532,13

1479,18

1424,16

1381,11

1354,17

1285,291251,28

1228,19

1186,311171,33

1045,14

993,33

939,36914,37

870,34

789,21

751,35

704,28642,28

572,37

541,40505,39

470,30

(a)




937


Sailee A B-10.sp - 5/19/2015 - TSI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-3.0

0

5

10

15

20

25

30

35

40

45

50

55

57.3

cm-1

%T

3506,14

3439,22

3230,73102,6

3062,6

2968,72937,8

2881,112831,12

2768,14

2035,47

1658,1

1613,7

1593,181532,18

1506,21

1480,191422,18

1400,18

1381,11

1352,22

1310,33

1284,361250,35

1229,23

1185,391168,39

1088,45

1071,34

1045,13

996,40

971,42941,42

867,40

817,47

776,30

751,36682,38

659,39

643,38

598,43572,41

540,45504,45

461,39

430,44

Fig. 3 FTIR spectra for (a) Thiamine hydrochloride (unirradiated)

(b) Thiamine hydrochloride (irradiated)


Sailee A B-11.sp - 5/19/2015 - BFU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-2.0

0

2

4

6

8

10

12

14

16

16.8

cm-1

%T

3525,2

3369,1

2916,2

1677,01614,0

1497,6

1396,1

1341,3

1277,51249,5

1223,61200,5

1112,1

1077,21042,2

1028,2

988,6

869,10842,10

821,9

758,6702,6

672,8644,8

625,7598,7

576,7

527,7

470,11446,11

415,13

407,14

(b)

(c)




938


Sailee A B-12.sp - 5/19/2015 - BFI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

3.0

4

6

8

10

12

14

16

18

20

22

24

26

28

30.0

cm-1

%T

3692,22

3677,22

3370,6

2916,13

1677,91619,9

1422,13

1397,11

1342,16

1279,201246,20

1201,20

1113,101068,11

1029,9

990,15

943,24913,25

868,26846,27

828,27

757,22

702,20669,20

644,21

624,20576,19

535,18

468,19

450,20

Fig. 3 FTIR spectra for (c) Beplex Forte (unirradiated)

(d) Beplex Forte (irradiated)


Sailee A B-13.sp - 5/19/2015 - NFU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

0.0

2

4

6

8

10

12

14

16

18

20

22

24

26.9

cm-1

%T

3367,2

2932,72882,8

1732,13

1678,21656,3

1620,41592,3

1577,31551,3

1423,41400,4

1369,6

1341,7

1308,11

1270,13

1254,11

1202,10

1154,51124,5

1077,31029,2

936,16917,16

887,17862,18

828,18

806,19

774,16

703,13

671,19

644,17

623,13

576,8

532,12

448,23411,22

(d)

(e)




939


Sailee A B-14.sp - 5/19/2015 - NFI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

7.0

8

10

12

14

16

18

20

22

24

26

28

30

32

34.4

cm-1

%T

3366,11

2931,18

2882,19

1732,24

1677,121656,12

1620,15

1577,121551,12

1424,141400,15

1369,17

1342,18

1308,221254,22

1201,21

1154,151124,16

1077,12

1040,101029,10

862,27828,28

773,27

703,25

670,28

573,19

532,22

448,31

410,32

Fig. 3 FTIR spectra for (e) Neurobion Forte (unirradiated)

(f) Neurobion Forte (irradiated)


Sailee A B-15.sp - 5/19/2015 - AAU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-3.0

-2

0

2

4

6

8

10

12

14

16

18

20

22

24

25.0

cm-1

%T

3945,22

3526,13411,0

3317,1

3218,13031,0

2916,1

2744,1

2043,20

1754,1

1673,0

1497,41456,4

1388,31363,2

1321,01275,1

1249,21221,2

1198,2

1140,01121,0

1076,21067,2

1044,21026,0

988,2

869,6

821,4

756,2

720,5682,4

628,7

592,13

565,6

493,19

471,16

447,12

(f)

(g)




940


Sailee A B-16.sp - 5/19/2015 - AAI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-3.0

0

5

10

15

20

25

30

34.8

cm-1

%T

3526,4

3411,33316,4

3218,5

3029,32916,4

2744,6

2043,32

1754,4

1673,1

1497,101455,9

1388,7

1363,6

1321,1

1275,3

1248,61221,6

1198,6

1140,21121,1

1076,51067,5

1044,5

1026,1

988,4

869,10

821,8

756,5

721,9682,8

628,11

592,17

565,10

493,21

472,19

448,15

Fig. 3 FTIR spectra for (g) Ascorbic acid (unirradiated)

(h) Ascorbic acid (irradiated)


Sailee A B-17.sp - 5/19/2015 - CEU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

0.0

2

4

6

8

10

12

14

16

18

20

22

24

26

28.7

cm-1

%T

3527,7

3412,63317,6

3219,7 3031,62917,6

2742,7

1754,6

1674,3

1497,91458,8

1388,71363,6

1321,4

1275,5

1249,71221,7

1198,61141,4

1121,4

1076,61067,6

1044,6

1026,4

989,6

869,10

821,9

756,6

721,9683,9

628,11

592,14

566,10

493,18

472,16

448,14

(h)

(i)




941


Sailee A B-18.sp - 5/19/2015 - CEI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-2.0

0

2

4

6

8

10

12

14

16

18

20

22

24

26

27.4

cm-1

%T

3527,33411,2

3317,3

3218,33031,2

2916,3

2851,42743,4

2043,25

1754,3

1673,0

1497,81456,7

1388,5

1363,4

1321,1

1275,2

1248,41221,5

1198,4

1140,11120,1

1076,31067,4

1044,3

1026,1

988,3

869,9

821,7

756,4

720,8

681,7

628,9

591,13

565,8

493,17

471,14

447,12

Fig. 3 FTIR spectra for (i) Celin (unirradiated) (j) Celin (irradiated)


Sailee A B-19.sp - 5/19/2015 - LMU

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-1.0

0

2

4

6

8

10

12

14

16

18

20

22

24

25.9

cm-1

%T

3563,5

3388,13322,1

3013,102993,10

2970,8

2916,5

2850,8

2724,132646,13

1892,22

1702,7

1600,4

1460,61429,6

1386,8

1363,61347,6

1321,4

1277,6

1233,7

1209,10

1158,5

1127,21070,1

1050,1

1013,31003,3

990,2

940,9

909,7

867,11

849,13

829,17

753,10722,10

694,10654,9

601,9583,8

551,10

537,12

521,13

496,17

470,18

425,19

406,24

(j)

(k)




942


Sailee A B-20.sp - 5/19/2015 - LMI

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

-2.0

0

2

4

6

8

10

12

14

16

17.9

cm-1

%T

3971,16

3563,2

3323,0

3013,5

2916,2

2850,3

2724,62648,7

1887,14

1703,3

1599,1

1460,21428,2

1386,31363,2

1347,21321,1

1277,2

1233,3

1209,5

1127,01069,0

1050,0

989,0

940,4

909,3

867,6

849,7

829,10

753,5722,5

694,5653,4

601,5583,4

550,5

537,6

521,7

496,10

471,11

423,12

Fig. 3 FTIR spectra for (k) Limcee (unirradiated) (l) Limcee (irradiated)

The NMR spectra of unirradiated and irradiated Thiamine hydrochloride, Beplex Forte,

Neurobion Forte, ascorbic acid, Celin and Limcee are shown in Fig. 4 (a-l).

In the NMR spectra of thiamine hydrochloride (Fig. 4 a and b) two singlets are observed at δ

2.4801 and δ 2.546 ppm, each having an intensity 3 corresponding to the two methyl groups

at C13 and C18 position in thiamine. The triplets observed at δ 3.1212 and δ 3.809 ppm

correspond to C14 and C15 CH2 protons respectively having intensity approximately 2. The

singlet peak at δ 5.5091 represents the CH2 protons at C6 position with intensity 2 and this

confirms that there is no deformation or splitting in the structure due to gamma radiation.

The highly deshielded peaks observed at δ 7.9781 and δ 9.607 ppm correspond to the

aromatic protons and the singlet at δ 4.6992 ppm corresponds to the hydroxyl proton.

(l)




943

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2.4801

2.5646

3.1068

3.1212

3.1357

3.7944

3.8090

3.8234

4.6992

5.5091

7.9781

9.6070

3.11

3.00

2.05

2.05

2.09

1.00

0.71

Current Data Parameters

NAME May11-2015

EXPNO 320

PROCNO 1

F2 - Acquisition Parameters

Date_ 20150511

Time 18.27

INSTRUM spect

PROBHD 5 mm PABBO BB-

PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 90.5

DW 41.600 usec

DE 6.00 usec

TE 297.2 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz

F2 - Processing parameters

SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

TSUBRUKER

AVANCE II 400 NMR

Spectrometer

SAIFPanjab University

Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2.4787

2.5646

3.1072

3.1216

3.1360

3.7964

3.8110

3.8253

4.6999

5.5078

7.9717

9.6049

3.06

3.02

2.05

2.08

2.08

1.00

0.74


NAME May11-2015

EXPNO 330

PROCNO 1


Date_ 20150511

Time 18.35

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 101

DW 41.600 usec

DE 6.00 usec

TE 297.4 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

TSIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig. 4 NMR spectra for (a) Thiamine hydrochloride (unirradiated)

(b) Thiamine hydrochloride (irradiated)

(a)

(b)




944

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.7762

0.8112

2.2878

2.3049

2.3221

2.3377

2.4255

3.2629

3.2908

3.3091

3.3262

3.3433

3.3804

3.4084

3.5250

3.5862

3.6031

3.6137

3.6243

3.6320

3.6387

3.6531

3.6679

3.8691

3.8877

3.8909

3.9056

3.9198

3.9237

4.3855

4.3880

7.3571

7.3695

7.3766

7.3894

7.4265

7.4391

7.4464

7.4591

8.0788

8.0985

8.1154

8.4563

8.4686

8.5552

8.5676

8.7725

8.7767

8.7926

1.17

2.06

13.62

2.05

1.19

0.37

1.04

1.19

0.32

0.87

1.00


NAME May11-2015

EXPNO 341

PROCNO 1


Date_ 20150511

Time 18.45

INSTRUM spect


PULPROG zgpr

TD 32768

SOLVENT D2O

NS 16

DS 2

SWH 12019.230 Hz

FIDRES 0.366798 Hz

AQ 1.3631988 sec

RG 114

DW 41.600 usec

DE 6.00 usec

TE 297.5 K

D1 5.00000000 sec

d12 0.00002000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

PL9 50.23 dB

SFO1 400.1318806 MHz


SI 16384

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.00 Hz

GB 0

PC 1.00

BFUBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.7896

0.8246

2.2992

2.3163

2.3334

3.2761

3.3043

3.3212

3.3382

3.3460

3.3553

3.3699

3.3935

3.4217

3.4392

3.4488

3.4640

3.4738

3.5749

3.6001

3.6137

3.6182

3.6244

3.6352

3.6426

3.6497

3.6641

3.6886

3.7081

3.7161

3.7230

3.7665

3.8803

3.8968

3.9014

3.9115

3.9158

3.9192

3.9311

3.9530

3.9741

4.1054

4.1273

4.3977

4.4025

4.7015

5.3052

5.3148

7.4484

7.4606

7.4683

7.4806

8.0970

8.1020

8.1062

8.1167

8.1210

8.1263

8.5745

8.5781

8.5867

8.5905

8.7969

8.8013

8.8111

8.8154

0.85

0.82

0.77

2.10

0.95

2.78

1.76

0.84

1.83

0.42

1.00

0.43

0.24

0.82

1.03

0.25

0.82

1.07


NAME May11-2015

EXPNO 350

PROCNO 1


Date_ 20150511

Time 18.50

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 287

DW 41.600 usec

DE 6.00 usec

TE 297.6 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

BFIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig. 4 NMR spectra for (c) Beplex Forte (unirradiated)

(d) Beplex Forte (irradiated)

(c)

(d)




945

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.8020

0.8374

0.8582

2.3170

2.3341

2.3511

2.3660

2.3859

2.4014

2.4512

2.4620

2.4675

3.0791

3.0943

3.2885

3.3165

3.3365

3.3536

3.3706

3.4056

3.4336

3.7645

3.7684

3.7830

3.7976

3.8938

4.6349

4.7023

4.7282

5.3366

7.4589

7.4709

7.4784

7.4910

7.9279

8.1063

8.1111

8.1159

8.1264

8.1311

8.1360

8.5846

8.5883

8.5970

8.6008

8.8030

8.8080

2.27

2.16

1.89

3.00

0.84

1.10

1.05

1.06

1.02


NAME May11-2015

EXPNO 360

PROCNO 1


Date_ 20150511

Time 18.58

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 322

DW 41.600 usec

DE 6.00 usec

TE 297.7 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

NFUBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.8036

0.8390

0.8597

2.3214

2.3384

2.3554

2.3749

2.3930

2.4631

3.0815

3.0963

3.2900

3.3181

3.3391

3.3561

3.3731

3.4071

3.4352

3.7852

3.8954

4.7024

4.7373

5.3431

7.4609

7.4622

7.4742

7.4811

7.4823

7.4931

7.4943

7.9292

8.1089

8.1141

8.1188

8.1290

8.1342

8.1388

8.5866

8.5904

8.5992

8.6029

8.8054

8.8102

2.31

2.15

1.89

3.00

0.75

1.13

1.07

1.08

1.04


NAME May11-2015

EXPNO 370

PROCNO 1


Date_ 20150511

Time 19.05

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 203

DW 41.600 usec

DE 6.00 usec

TE 297.8 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

NFIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig. 4 NMR spectra for (e) Neurobion Forte (unirradiated)

(f) Neurobion Forte (irradiated)

In the NMR spectra of ascorbic acid (Fig. 4 g and h) the methylene CH2 protons are found in

the most deshielded region of NMR at δ 3.619 ppm with intensity 2. The methyne CH proton

gives multiplet split at δ 3.9417 ppm and the aromatic methyne proton gives singlet at δ

4.7107 ppm. The aliphatic hydroxyl proton is observed at δ 4.8334 ppm.

(e)

(f)




946

The NMR spectra for unirradiated and irradiated standards and tablets are almost identical

indicating that gamma irradiation does not cause any chemical change in the vitamins.

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.6163

3.6190

3.6341

3.9246

3.9292

3.9400

3.9417

3.9443

3.9572

3.9618

4.7107

4.8287

4.8334

2.03

1.00

1.01


NAME May11-2015

EXPNO 380

PROCNO 1


Date_ 20150511

Time 19.13

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 181

DW 41.600 usec

DE 6.00 usec

TE 297.9 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

AAUBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.6053

3.6081

3.6230

3.9133

3.9178

3.9292

3.9316

3.9333

3.9460

3.9505

4.7124

4.8155

4.8201

2.03

1.00

1.04


NAME May11-2015

EXPNO 390

PROCNO 1


Date_ 20150511

Time 19.21

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 161

DW 41.600 usec

DE 6.00 usec

TE 298.1 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

AAIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig 4: NMR spectra for (g) Ascorbic acid (unirradiated) (h) Ascorbic acid (irradiated).

(g)

(h)




947

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.6224

3.6252

3.6401

3.9307

3.9352

3.9465

3.9507

3.9634

3.9680

4.7102

4.8334

4.8379

2.08

1.00

1.08


NAME May11-2015

EXPNO 400

PROCNO 1


Date_ 20150511

Time 19.28

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 181

DW 41.600 usec

DE 6.00 usec

TE 298.2 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

CEUBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.6432

3.6590

3.9497

3.9513

3.9541

3.9647

3.9667

3.9690

3.9706

3.9820

3.9834

3.9865

4.7082

4.8554

4.8572

4.8599

4.8614

2.10

1.01

1.00


NAME May11-2015

EXPNO 410

PROCNO 1


Date_ 20150511

Time 19.36

INSTRUM spect


PULPROG zg30

TD 65536

SOLVENT D2O

NS 8

DS 2

SWH 12019.230 Hz

FIDRES 0.183399 Hz

AQ 2.7263477 sec

RG 144

DW 41.600 usec

DE 6.00 usec

TE 298.3 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

SFO1 400.1324710 MHz


SI 32768

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

CEIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig. 4 NMR spectra for (i) Celin (unirradiated) (j) Celin (irradiated)

(i)

(j)




948

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.3581

3.3816

3.4051

3.4547

3.4641

3.4797

3.4892

3.5890

3.6139

3.6326

3.6428

3.6523

3.6613

3.6673

3.6747

3.6981

3.7298

3.7594

3.7670

3.7789

3.7890

3.8006

3.8102

3.8157

3.8254

3.9249

3.9412

3.9617

3.9832

4.1155

4.1373

4.5096

5.3178

5.3272

1.12

1.15

2.23

3.76

6.43

2.50

1.00

1.04

1.03


NAME May11-2015

EXPNO 421

PROCNO 1


Date_ 20150511

Time 19.46

INSTRUM spect


PULPROG zgpr

TD 32768

SOLVENT D2O

NS 16

DS 2

SWH 12019.230 Hz

FIDRES 0.366798 Hz

AQ 1.3631988 sec

RG 71.8

DW 41.600 usec

DE 6.00 usec

TE 298.5 K

D1 5.00000000 sec

d12 0.00002000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

PL9 50.23 dB

SFO1 400.1318806 MHz


SI 16384

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.00 Hz

GB 0

PC 1.00

LMUBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.3555

3.3790

3.4024

3.4524

3.4619

3.4772

3.4867

3.5865

3.6109

3.6295

3.6400

3.6498

3.6585

3.6645

3.6724

3.6960

3.7268

3.7561

3.7639

3.7763

3.7864

3.7978

3.8070

3.8126

3.8228

3.9241

3.9388

3.9589

3.9804

4.1127

4.1345

4.5064

4.5088

5.3145

5.3240

1.00

1.04

2.09

3.65

6.34

2.49

1.04

1.01


NAME May11-2015

EXPNO 431

PROCNO 1


Date_ 20150511

Time 19.54

INSTRUM spect


PULPROG zgpr

TD 32768

SOLVENT D2O

NS 16

DS 2

SWH 12019.230 Hz

FIDRES 0.366798 Hz

AQ 1.3631988 sec

RG 90.5

DW 41.600 usec

DE 6.00 usec

TE 298.4 K

D1 5.00000000 sec

d12 0.00002000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1H

P1 10.90 usec

PL1 -3.00 dB

PL9 50.23 dB

SFO1 400.1318806 MHz


SI 16384

SF 400.1300000 MHz

WDW EM

SSB 0

LB 0.00 Hz

GB 0

PC 1.00

LMIBRUKER

AVANCE II 400 NMR

Spectrometer


Chandigarh

[email protected]

Fig. 4 NMR spectra for (k) Limcee (unirradiated) (l) Limcee (irradiated)

Radiolysis of thiamine hydrochloride

The absorption spectra of irradiated thiamine hydrochloride solution at pH 6.8 with

characteristic absorption maxima at 232 and 266 nm, as recorded on Elico SL-210 double

beam UV-visible spectrophotometer, are shown in Fig. 5.

(k)

(l)




949

Fig. 5 Absorption spectra of thiamine hydrochloride aqueous solution.

Fig. 6 shows a plot of G(-TH) against the absorbed dose for aqueous thiamine hydrochloride

solution.

Fig. 6 A plot of G(-TH) against absorbed dose for aqueous thiamine hydrochloride

solution.

Thiamine hydrochloride was found to be resistant to gamma radiations in the solid state as

hardly any change was observed in the absorbance after irradiation till 50 kGy. However, it

undergoes extensive degradation in aqueous state and gets completely degraded after 1 kGy.




950

Radiolysis of ascorbic acid

Ascorbic acid in the solid state was highly resistant to gamma radiations and there was no

variation in the amount of ascorbic acid in the tablets after irradiation till 50 kGy. Also, in

aqueous solution, ascorbic acid was found to be resistant to much higher gamma dose as

compared to thiamine.

Fig. 7 (a and b) shows the amount of ascorbic acid remaining and G(-AA) against the

absorbed dose. The results of tablets, Celin and Limcee, have been compared with standard

ascorbic acid.

Fig. 7 (a) A plot of amount of ascorbic acid remaining

(b) G(-AA) against absorbed dose for ascorbic acid and tablets, Celin and Limcee

CONCLUSION

i) Radiation sterilization of water soluble vitamins can be effectively carried out in solid

state as decomposition of vitamins does not occur due to gamma irradiations in the solid

state.

ii) Irradiation does not cause any change in the chemical structure too, as is evident from

FTIR and NMR spectra.

iii) Aqueous solution of thiamine hydrochloride undergoes extensive damage due to

gamma irradiation.




951

iv) Aqueous solution of ascorbic acid is quite resistant to gamma radiation and does not

undergo much radiation damage at low dosages of gamma irradiation. Thus, vitamin C

syrups can also be sterilized by low dose of gamma radiation.

ACKNOWLEDGEMENTS

I am thankful to Head, Department of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur

University, for offering me access to laboratory, instruments and gamma irradiation facilities.

Funding

I am gratified to University Grants Commission, New Delhi for awarding Rajiv Gandhi

National Fellowship for carrying out my research work.

REFERENCES

1. Reid BD. (Gamma processing technology, an alternative technology for terminal

sterilization of parenterals). PDA J Pharm Sci Tech, 1995; 49(2): 839.

2. Abuhanoğlu G, Özer AY. (Radiation effects on pharmaceuticals). FABAD J Pharm Sci,

2010; 35: 20317.

3. Jacobs GP, Wills PA. (Recent developments in the radiation sterilization of

pharmaceuticals). Radiat Phys Chem, 1988; 31: 68591.

4. Colovos G, Churchill BW. (The electron beam sterilization of certain pharmaceutical

preparations). J Pharm Assoc Sci Ed, 1957; 46: 5803.

5. Rhee KS, Chun KJ, Kim KS. (Radiation sterilization of medical products (II), effect of

gamma irradiation Co-60 on tetracycline hydrochlorides). Misaengmul Hakhoe Chi,

1975; 13: 6470.

6. Kishore K, Moorthy PN, Rao KN. (Study of radiation damage and protection of some

vitamins in aqueous media). Proc Indian natn Sci Acad, 1976; 42B(4-5): 23849.

7. Kishore K, Moorthy PN, Rao KN. (Radiation protection of vitamins in aqueous systems).

Radiat Eff, 1976; 27: 16771.

8. Kishore K, Moorthy PN, Rao KN. (Radiation protection of vitamins in aqueous systems,

Part II). Radiat Eff, 1976; 29: 16570.

9. Kishore K, Moorthy PN, Rao KN. (Radiation protection of vitamins in aqueous, systems,

Part III). Radiat Eff, 1978; 38(1-2): 97105.

10. Wilska-Jeszka J. (Comparative studies on the resistance of group B vitamins to gamma

irradiation). Nucleonika, 1977; 22: 10119.




952

11. Hacișevki A. (An overview of ascorbic acid biochemistry). J Fac Pharm Ankara, 2009;

38(3): 23355.

12. Rao BSN. (Radiolysis of ascorbic acid in aqueous solution by gamma radiation). Radiat

Res, 1962; 17(5): 68393.

13. L’ova MS, Belkina NP, Nozlova EI. (Action of gamma-irradiation of solutions of

ascorbic acid for injection). Khim Farm Zh, 1980; 14: 814.

14. Rao CNR. (Chemical Applications of Infrared Spectroscopy). Academic Press, New

York, 1963.

15. Wilk IJ. (Problem-causing constituents of vitamin C tablets). J Chem Educ, 1976; 53(1):

413.


GAMMA RADIOLYTIC DEGRADATION OF VITAMIN B1 AND …

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