STABILIZATION OF AQUEOUS SOLUTIONS OF RIBOFLAVIN S. Astanov, A. S. Prishchepov, and E. K. Pil'ko

B. D. Zaripov, UDC 535.33/34

In natural in vivo conditions, riboflavin is synthesized by the intestinal microflora; its deficiency in the human organism leads to skin diseases, loss of vision, and chronic gastritis and colitis. Riboflavin is basic to many medical formulas, which are sterilized by thermal means. This vitamin is also present in many fish products. In connection with the light conditions during the production and storage of riboflavin preparations and with its sterilizational heat treatment, it is of interest to investigate its thermo- and photostability and to develop nonchemical methods of stabilizing the vitamin. Commercial riboflavin powder is used in the present work [i]. Riboflavin solutions are prepared from distilled water in concentrations of 2.10-s-2.10 -4 g/ml. For complete solution of the preparation, it is heated briefly to 323 K, after which the solution is passed through a glass filter. Powders and solutions of the vitamin are subjected to accelerated aging by heat treatment: solutions in a thermostat at 343 K in tightly sealed cuvettes for 34 hr and by boiling in normal conditions; and powder in a desiccator for i hr at 371, 393, 453, 463, and 473 K. In addition, for accelerated aging of the preparation, investigation of its optical strength, and possible stabilization, aqueous solutions of the vitamin are irradiated by light from an LPM-II helium-cadmium laser with a radiation wavelength of 441.6 nm and a power of 30 mW for 20 hr, and also by light from an LG-126 helium-neon laser with a wavelength of 632.8 nm and a power of 80 mW for 1-8 hr. Accelerated aging of the powder is also undertaken by IR irradiation using an ILGN-701 carbon-dioxide laser with a radiation wavelength of 10,600 nm and a power of 60 W through a scattering germanium lens for 2-7 sec. The change in structure of the riboflavin in the laser and heat treatment of its powders and solutions and also after prolonged (up to 1.5 months) storage of its solutions is monitored by the methods of absorptive spectroscopy, luminescence, magnetic resonance, and dispersion of optical rotation; the aggregate state of the preparation is monitored using linear-dichroism spectroscopy. The first four methods here are used to establish the native character of the vitamin and linear dichroism to determine the degree of its solution in medicinal form and the solution before and after thermal and optical treatment. The absorption spectra in the visible and UV regions are recorded using a Beckman UV5270 spectrophotometer. The IR transmission spectra are recorded on a UR-20 spectrometer. The optical-rotation dispersion spectra are recorded on a Spectropol-i spectropolarimeter. The linear dichroism is monitored on a Jasco-20 circular dichrograph modified using an optical attachment. The luminescence spectra are recorded on a Fica-55 spectrofluorimeter, and the NMR spectra on a WM-360 spectrometer (Bruker). The IR transmission of the initial and treated riboflavin powder is measured after preliminary pressing into tablet form with KBr filler (300 mg KBr 2 per 2 mg of dry powder). The proton resonances of riboflavin are recorded in deuterated dimethylsulfoxide (CD3SOCD3), in which the vitamin is dissolved to concentrations considerably exceeding its limiting concentration in an aqueous medium. To eliminate reabsorption of the luminescence in the volume of the riboflavin solution, solutions with an optical density no greater than 0.i at the maximum of the absorption band at a wavelength of 440 nm are prepared. The absorption, luminescence, IR transmission, and NMR spectra of freshly prepared riboflavin samples, characterizing the native untreated preparation, are shown in Fig. i. In the given IR transmission spectra, in the spectral ranges 400-1900 and 2600-3900 cm -I, bands typical for the riboflavin molecule corresponding to valence C--H and C=<) vibrations, C=N vibrations in the heterocycle chain, valence N-H vibrations, deformational N-H vibrations, and also O-H vibrations are observed. In the NMR spectrum, in the region of chemical shifts

Belorussian State Institute of the Development of Doctors, Ministry of Public Health, USSR. B. I. Stepanov Physics Institute, Academy of Sciences of the USSR, Minsk. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 53, No. i, pp. 75-82, July, 1990. Original article submitted July 4, 1989. 0021-9037/90/5301-0735512.50

9 1991 Plenum Publishing Corporation

735

ilum rel. units

:'i

b d

l

O' ~,

q

q

]

q~

d ki''!i'! I :i

i

~X\~

/ x'x.~'J ~ cm-~

'~ i iV r

I~OD 2680

?00

3FO0

0~3

J

0,~ J

l /k/ |/

O I

,

,

~, 1 `\72 \/ \

i

}%

IZ

~ /, ~

h~ ~I

,i'~ . . . . . . . . . . . . . . . .

7/

~350CD~

/

r-" ~/ - -\ ~-v - = ,~ 208 YOO qnO S!d: 6Pq 1 um

6, p.p.m

c

7L?

~

E

7

~

ii, I i

' I~

~

I

/

I . . . .

~

~

!! :I

;, !

. I

3

!L

I . . . . . .

2

I

0

Fig. i. Absorption (i) and luminescence (2) spectra (a), IR transmission spectrum (b), and NMR spectrum (c) of riboflavin in aqueous solution (a), in KBr (b), and in CD3SOCD 3 ( c ) . 3-12 p.p.m, proton resonances of the groups CH3, O-H, G-H, CH2, and N-H are observed, as a doublet of opposed protons in the benzene ring. Table 1 gives the optical densities maxima of the absorption bands in the UV fore and after treatment of the solutions strong dilution and prolonged storage at

as well

of aqueous solutions of riboflavin at the basic and visible regions of the absorption spectra beby heating and optical radiation and also after 277 K.

A clearly expressed dependence of the amplitude of the UV absorption bands of riboflavin on its concentration in aqueous solution is seen; this is evidently a consequence of the partial weak aggregation of the preparation at high concentrations (2.10 -~ g/ml) and its disaggregation with reduction in concentration by dilution of the initial solution to 8.10 -6 g/ml: the amplitudes of the bands with maxima at wavelengths of 220 and 370 nm are more sensitive to the state of aggregation of riboflavin. With prolonged storage of the solution, the optical densities in these bands change - decrease and increase, respectively; in dilution of the solution the opposite picture is seen - increase in amplitude of the band with a maximum at a wavelength of 220 nm and decrease in amplitude of the band with a maximum at 370 nm. Prolonged heat treatment of riboflavin solution even at 343 K leads to change in its optical densities. At the same time, treatment of the powder at 371, 393, 453, and 463 K in a dessicator for 1 hr and treatment of dry powder by the thermal radiation of a carbon-dioxide laser for 2-3 hr leads to no pronounced change in absorption spectrum. Change in the absorption spectra is only observed after heat treatment of the preparation by hot air at 673 K, and also after 7-sec treatment by thermal laser radiation, which is not associated with change in the aggregate structure of the riboflavin solution in comparison with that of the initial solutions of untreated preparation. These changes are due to thermodestruction. In fact, after vacuum evaporation of water from heat-treated solutions of the vitamin, riboflavin powder is obtained and, as for the heat-treated powder, its NMR and IR transmission spectra are recorded: the spectra of freshly prepared samples and preparations treated in "mild" conditions coincide (Fig. i); change in these spectra is seen only at a treatment temperature of ~73 K and after 7-sec treatment of riboflavin power by a CO z laser. In the irradiation of aqueous solutions of riboflavin by light from a helium-cadmium laser, which is effectively absorbed by the preparation, a redistribution of the absorptionband amplitudes is seen, together with a tendency to general reduction in amplitude by 1-14~, depending on the specific band. The bands which are most sensitive to optical treatment of the preparation are those with maxima at 220 and 265 nm (13 and 14~ change in amplitude, respectively); the least sensitive are those with maxima at 370 and 4&O nm (I and 7Z decrease in amplitude). There is also significant change in the NMR and IR transmission spectra here, as a result of destruction of the preparation and its decoloration due to breakdown into urea

736

TABLE I. Optical Density (D) of Aqueous Solutions of Riboflavin at the Maxima of the Absorption Bands (%max) Solution Freshly prepared

Xmax

T, K 440

370

265

250

0,568 0,499

1,75 1,62

1,62 1,50

393

I

D

After prolonged storage

281

0,653 0,57

Initial freshly prepared soln. after 25-fold dilution (9,= 1 am)

393

0,261

0,211

0,714

0,754

After heat treatment

343 371

0,527 0,653

0,54 0,568

1,65 1,75

1,64 1,62

393 453 463 473

0,653 0,653 0,653 0,62 0,653

0,568 0,568 0,568 0,531 0,568

1,75 t,75 1,75 1,62 1,75

1,62 I ,62 1,62 1,58 1,62

0,64

0,557

1,72

1,63

393

0,61

0,562

1,496

I ,4i

393

0,653

0,568

1,75

1,62

After heat treatment of 9 the powder

)> ))

After treat, of the powder by by a CO2 laser (2-3 sec) After treat, of the powder by a CO2 laser (7 sec) After treat, of freshly prepared solution by a helium-cadmium laser After treat, of freshly prepared solution by a he litmmmeon laser

Note. The error in measuring the optical densities is 3-5%. The cuvette thickness is 0.i cm. After thermal and laser treatment of the powder, it is dissolved in distilled water, and measurements are made at 393 K.

and 1,2-dihydro-6,7-dimethyl-2-keto-l-ribityl-3-quinoxaline carbolic acid [2]. Irradiation of the aqueous solutions by light from a helium-neon laser with a wavelength of 632.8 nm does not lead to change in spectral-optical characteristics of the preparation. Preparation of a medicinal form of riboflavin in practice includes its solution of the powder in water for injection, until a 0.02% solution is obtained, and sterilizational heat treatment of the resulting solution by steam at 373 K for 30 min. It is widely used in medical practice, for example, as vitamin eye drops and concentrates for the preparation of eye drops. However, as shown by investigation, riboflavin is partially broken down when aqueous solutions are subjected to heat treatment, and this means that the medicinal form obtained on this basis does not contain 0.02% riboflavin, but is reduced by 7.5% (calculated, as usual, from the optical density in the absorption band with a maximum at 265 nm [i]). In addition, the quality of the medicinal form is low because of the presence of destruction products of the preparation. As follows from the present experiment, this may be prevented by separate sterilizational heat treatment of the riboflavin powder and the distilled water, with subsequent mixing in aseptic conditions. The powder may be heat treated using hot air with a temperature no greater than 453-463 K or with short-term treatment by the thermal radiation of a CO 2 laser. Since the heat treatment of the riboflavin powder is possible by hot air at 453-463 K or thermal laser radiation with no thermodestruction of the preparation, separate sterilizational heat treatment of the riboflavin powder and the water with subsequent mixing in aseptic conditions allows a stable medicinal form of the vitamin to be obtained without loss of the basic active ingredient in solution and without the introduction of destruction products. Linear-dichroism spectroscopy is a very informative and accurate method of monitoring the degree of aggregation of riboflavin in aqueous solution. Since the aggregates of the vitamin are oblong microcrystals, the preparation becomes optically anisotropic on incomplete solution or aggregation to the colloidal state in storage, in conditions where these solutions

737

run through a flow-through cuvette, on account of the partial orientation of the riboflavin aggregates in the hydrodynamic laminar flow. In this case, a linear dichrograph recorded a curve differing from the zero line: a linear-dichroism spectrum with a band maximum corresponding to the maximum of the absorption band of the aggregated riboflavin. The amplitudes of the linear-dichroism bands are proportional to the degrees of aggregation of the riboflavin solutions. The possibility, in principle, of measuring linear dichroism was predicted in [3-5]. One possibility may be realized on the basis of a Jasco-20 circular dichrograph, using an optical attachment designed and constructed for the visible and UV spectral regions: a double Fresnel parallelepiped. In practice, a suitable measure of the circular dichroism is the ellipticity determined by the ratio of the minor and major axes of the ellipse [6] tg 0 --~ 0 = tg ~

for small Q, where •

(ZL - - Z~) l ~--33Cl

(8 L --

8R) ,

(1)

XR, eL, ER are the absorption coefficients and molar extinction co-

efficients of the material for light with wavelength I of left (L) and right (R) circular polarization; C is the concentration of the optically active material; s is the same thickness. It is known that 1

--IOgloIR/IL, Cl

AS~-gL--eR =

(2)

where IR, I L are the intensities of the light transmitted by the sample with right and left circular polarization. Hence 0 = 33 IOgloIR/fL = 33 ]Oglo 1 + 12/2I1 ,

1 where

Ii

= (1/2)

(I R + IL),

12 = I R -

I L,

-

-

(3)

12/211

by definition.

Since for small optical activities

IL [R-~ IL

[R --

I.~

~--<<

211

1,

(4)

it follows that

1

-

-

12/2I~

~" . . . .

Io

e-"~"~"

fz

log~o e.

(5)

Hence

(6)

e = 3 3 / _ L log,,e, ll

i.e., the ellipticity (in degree) is proportional to the ratio of the intensities I2, 11 recorded by the photoelectric system of the circular dichrograph. On transition from circular (CD) to linear (LD) dichroism, formula for 8 in a different form ~CD : 28.6

[R--IL IR + IL

it is convenient to use the

(7)

When circular polarization is incident on a Fresnel double parallelepiped, it is transformed into linear polarization; for R and L circular polarizations, the corresponding linear polarizations are orthogonal. Hence, a circular dichrograph measuring 8CD (deg) fitted with an optical attachment in the form of a Fresnel double parallelepiped positioned b e h i n d t h e Pockels electrooptical cell and in front of the given sample measures the linear dichroism

738

? I

;

I I i'

6. i! :": - -g C

~J

i

_j~

tt

" ~ I

i

0 P:mm---v,-~..

eoo \ i

_2]

2

,-.:, '\- . . " i /

\t I7 .... " -

-,"-~-,

v

,"

/

".

..,

XX

-r-----~--

V

Fig. 2. Absorption (i) and linear-dichroism (2) spectra of an aggregated aqueous solution of riboflavin in conditions of laminar hydrodynamic flow. OLD

.,~11m--I

28,o:

:'

I

! l ~ + l~i

(8)

I'

where f• , I, are the intensities of the linearly polarized light transmitted through the sample and incident on the dichrograph photoreceiver I!.--I,:

lO-n: --I0 o~

e D - log e

12 -b ,sii

10 -D

e ->-.- b~elO< _~_ (, D : lo,>,,1 0,

-,- I0-SJ;I

: : th [ - ~ ( D , - - D : ) l o g < , t O ] ~

0

--

o--D

log e]

0

(D! ~D!).log<, 10

(9)

for small values of the argument. Thus, the linear dichroism measured by the dichrograph ADi. if= The l i m i t i n g sensitivity

sensitivity in linear

of the circular

dichroism:

Di--D~! dichrograph

ADi, lj~3-10-5

is

(?~D

(i0)

32.98

8CD = 10 -3 d e g ' c m -1 c o r r e s p o n d s

to its

units.

The a b s o r p t i o n and l i n e a r - d i c h r o i s m s p e c t r a o f an a g g r e g a t e d c o l l o i d a l s o l u t i o n o f r i b o f l a v i n r u n n i n g t h r o u g h a 1-mm f l o w - t h r o u g h c u v e t t e a t a r a t e o f 2 mm/sec a r e shown in F i g . 2. The v e l o c i t y v e c t o r o f t h e l a m i n a r h y d r o d y n a m i c f l u x i s o r i e n t e d a t a n e a r - 4 5 ~ a n g l e t o t h e polarization v e c t o r o f t h e l i n e a r l y p o l a r i z e d l i g h t i n c i d e n t on t h e c u v e t t e . With a g i n g o f the aqueous solutions of riboflavin, the tendency of the preparation to aggregation increases, as i n d i c a t e d by t h e a p p e a r a n c e and g r o w t h o f l i n e a r d i c h r o i s m i n t h e s e s o l u t i o n s i n c o n d i t i o n s o f f l o w t h r o u g h a c u v e t t e , a n a l o g o u s t o s p e c t r u m 2 i n F i g . 2. At t h e same t i m e , s o l u t i o n s o b t a i n e d f r o m r i b o f l a v i n powder t h a t i s n o t d e s t r o y e d by h e a t t r e a t m e n t and C 0 2 - 1 a s e r t r e a t ment r e t a i n t h e i r n a t i v e s t a t e much l o n g e r , w i t h o u t a g g r e g a t i o n . Linear dichroism is zero here. Data on the photochemical stabilization of the preparation obtained as a result of treating aqueous solutions of riboflavin with light from a helium-neon laser with a wavelength of 632.8 nm, for different energy densities and different specific powers per unit volume of solution, are also important. This optical radiation is not absorbed by the preparation itself. The conditions of laser irradiation of the riboflavin solution are shown schematically in Fig. 3; 1 is the integrating sphere with partially mat specular 2 and protective 3 coatings; 4 is the input aperture for the introduction of light beam 5 from heliumneon laser 6; 7 is the aperture for filling with an aqueous solution of riboflavin 8, and is closed by ground stopper 9, the internal face of which i0 has a curvature equal to that of the internal spherical surface of the sphere and is coated with a light-reflecting layer. Unirradiated and irradiated riboflavin solutions are placed in flasks that are transparent to visible light and are held in scattered solar light in room conditions. Table 2 gives 739

9\

6

Fig. 3.

Scheme of the irradiation of an aqueous riboflavin solution.

TABLE 2. Optical Density (D) of 0.02% Riboflavin Solution at the Maxima of the Absorption Bands (kmax) Irradiated solutions o

imx

i

~ o~ .

Freshly.pre- 220 pared 265 solutions i 370

O

energy density of laser radiation (Ared = 632.8 rim), J/m2 3- ItP i 9.10" .[ .1.107

radiation power per unit volume of solution, W/m ~ 7o :~o ,~{o [ 70 I 400 I 800 [ 70 400 ] 800 D

440

1,65 1,62 0,53 0,51

1,65 1,62 O, 53 0,51

1,65 1,62 0,53 0,5L

1,65 1,62 O, 53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 O, 53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

A week

220 265 37O i 440 !

l, 54 1,51 0,46 0,40

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

t ,65 I ,62 0 ,53 0 ,51

1,65 1,62 0,53 0,51

1,65 1,62 0,53 0,51

1,85 1,62 O, 53 0,51

1,65 1,62 0., 53 0,51

Two weeks

I 220

1,42

LI 265 370

1 ,o8

1,59 1,56 0,46 0,45

1,60 1,57 0,48 0,47

1,61 1,61 0,50 0,48

1,60 1,56 0,46 0,45

1 ,60 1 ,58 0 ,48 (3,47

1,61 1,61 0.50 0;48

1,60 1,56 0,46 0,45

1,60 1,57 0.48 O, 47

1,61

440

0,36 0,30

1,60 0,50 0,48

Note. The error in measuring the optical densities is 3-5%. Cuvette thickness 0.i cm; t is the holding time in light in room conditions. spectral characteristics of the state of these solutions before and after holding in the light; the solutions are preliminarily thermosterilized. I t follows from Table 2 that the optical density of the unirradiated riboflavin solutions decreases in the course of holding in scattered solar light, on account of photochemical destruction of the preparation. Thus, with holding for a week, the optical density of unirradiated solutions at a wavelength of 440 nm is reduced by 21 + 3%; after two weeks, t h e reduction is 40 + 3%. This indicates considerable photochemical destruction of the riboflavin. At the s~me time, riboflavin solutions irradiated by light from a helium-neon laser for a week with exposure to scattered solar radiation remain stable: the optical density of these solutions is unchanged. The IR luminescent, gyrotropic (riboflavin in water at neutral pH is optically inactive), and NMR characteristics of laser-irradiated solutions are also unchanged (Fig. i). Exposing the riboflavin solutions to light from a helium-neon laser with a power density of less than 3"108 J/m 2 and with a specific power per unit volume of the solution of 70 W/m 3 leads only to partial stabilization of the preparation: after two-week holding of these solutions in scattered solar light, their optical density at a wavelength of 440 nm decreases by 3-4%. Thermosterilized aqueous solutions of riboflavin with and without laser treatment are also subjected to the action of direct solar light. In this case the preparation in the unirradiated solutions begins to break down after only 8-h holding in the light. The irradiated solutions retain their optical-spectral properties for up to 1-2 days.

740

The reasons for the stabilization of aqueous riboflavin solutions by exposure to He-Na laser radiation are not totally clear. The action of radiation of wavelength 632.8 nm on cells and microorganisms is well known [7]. Riboflavin powder and aqueous solutions contain a series of aerobic bacteria, sporogenic bacilli, and yeast fungi [8] of dimensions 0.5-1.5 ~m, as well as viruses of size 0.02-1 ~m. In the present case, there is evidently additional sterilizing action of the He-Ne laser radiation on the microflora of the preparation in aqueous solution. LITERATURE CITED i, 2. 3. 4. 5. 6.

7. .

State Pharmacopoeia of the USSR [in Russian], Moscow (1968), p. 585. Pharmaceutical Inconsistencies [in Russian], Moscow (1965), p. 114. R. Mandel and G. Holzwarth, Rev. Sci. Instrum., 41, No. 5, 755-757 (1970). B. Norden, Chem. Scripta, 9, 49-50 (1976). A. Davidson and B. Norden, Chem. Scripta, ~, 167-168 (1975). L. Bellyuz, M. Legran, and M. Grozhan, Optical Circular Dichroism [in Russian], Moscow (1967). N. F. Gamaleya, E. D. Shishko, and Yu. V. Yanish, Izv. Akad. Nauk SSSR, Ser. Fiz., 50, No. 5, 1027-1029 (1986). I. V. Besedina, N. I. Bessonova, and V. V. Karchevskaya, Farmats., No. 3, 23-25 (1982).

IR SPECTRA AND STRUCTURE OF 2-OCH~- AND 3-OCH 34,6-O-BENZYLIDENE-~-METHYL-D-ALTROSIDES T. E. Kolosova, L. K. Prikhodchenko, and N. V. Ivanova

R. G. Zhbankov, UDC 535.33:543.42

IR spectroscopy is widely used in investigating the structural - including conformational - properties of hydrocarbons [i]. Low-temperature investigations expand the analytic possibilities of the method of IR spectroscopy, allowing valuable new information to be obtained on the conformational properties and specifics of inter- and intramolecular interactions [2]. In the present work, the IR spectra (400-3700 cm -l) of crystals of 2-OCH 3- and 3-OCH 3derivatives of 4,6-O-benzylidene-~-methyl-D-altrosides and their isotopically dilute analogs are investigated at T = 300 and 18 K. The objects of investigation are synthesized by the well-known method of [3]. O-deuterated analogs are prepared by several successive recrystallizations from CH3OD. The degree of substitution by deuterium is ~60%. Interpretation of the results obtained is based on the data of x-ray structural analysis of single crystals of these compounds obtained by slow crystallization from a solution in C2HsOH. The crystalline structure of derivatives of s-Daltrose was investigated in [4-6]. Benzylidene derivatives are of considerable interest as model compounds with a rigidly fixed conformation of the pyranose cycle. In the spectrum of 2-OCH3-4,6-O-benzylidene-a-methyl-D-altroside (Fig. i) at T = 300 K, there is one intense narrow band with a maximum at 3520 cm -I. With reduction in temperature, its position is unchanged. There is no significant increase in the intensity of this band. It may be concluded that the hydroxyl group in the given compound is included in an intramolecular hydrogen bond. The presence of a hydroxyl group at C-2 (3-OCH3-4,6-O-benzylidene-~-methyl-D-altroside) leads to the appearance of an intense band at 3475 cm -l in the spectrum. The sharp increase in intensity and shift to 3450 cm -I at T = 18 K indicates the participation of the OH group at C-2 in an intermolecular hydrogen bond. In the spectrum of 4,6-O-benzyiidene-~-methyl-DB. I. Stepanov institute of Physics, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 53, No. i, pp. 83-87, July, 1990. Original article submitted July 3, 1989.

0021-9037/90/5301-0741512.50

9 1991 Plenum Publishing Corporation

741