Utilization of charge-transfer complexation in the spectrophotometric determination of some monosaccharides through their osazone intermediates

0039-9140/87 $3.00 + 0.00 Pergamon Journals Ltd

Talanta,Vol. 34, No. 9, pp. 193-191, 1987 Printed in Great Britain

UTILIZATION OF CHARGE-TRANSFER COMPLEXATION IN THE SPECTROPHOTOMETRIC DETERMINATION OF SOME MONOSACCHARIDES THROUGH THEIR OSAZONE INTERMEDIATES MAGDA AYAD, SNED BELAL*, AFAF ABOLJ EL KHEIR and SOBHI EL AJIL Pharmaceutical Chemistry and Pharmaceutical Analytical Chemistry Department, College of Pharmacy, Zagaxig and Alexandria Universities, Zagaxig and Alexandria, Egypt (Received 21 February 1986. Revised 3 March 1987. Accepted 13 March 1987) Smmnary-Monosaccharide osaxones are utilized in the spectrophotometric determination of their parent compounds though charge-transfer complexation with two-electron acceptor reagents. The molar combining ratio and the optimum complexation conditions have been studied. The method has been used to analyse for glucose and fructose and in determining blood glucose.

Glucose and fructose are widely used in current medical practice as plasma substitutes and body fluid compensatory agents, and other hexoses such as galactose and mannose are of use as diagnostic aids. Several procedures have been reported for their assay in ampoules or biological fluids, including titrimetric,‘** potentiometric,3*4 polarographic,s fluoriatomic-absorption spectrophotometric,’ metric,6 spectrophotometric,g14 NMR,15 GC?‘* and HPLCLS2’ methods. Many of the calorimetric methods depend on the reducing action of the sugar molecule on chromogenic reagents such as the cupric ion,” silver salt~,*~mercury salt~,~~~ halogens in alkaline media,26 ferricyanide,2’ periodate,2* ceric salts,29 tetraxolium salts,M aromatic nitro-compounds,3’ phosphomolybdate,32 mineral acid,33 anthrone,” phenolic compounds in mineral acid,3S37 or aromatic amines.38 Condensation of hexoses with phenylhydraxine to form a hydraxone or an osaxone has also been utilir.ed3’ and claimed4 to give true values for glucose in biological fluids. Charge-transfer complexation reactions are now finding wide use in the determination of electrondonating nitrogenous bases, the reagents being the o-acceptor iodine”* or some II -acceptors (polyhaloor polycyanoquinones)4e52 in organic solvents. In earlier work,r3 we determined corticosteroid drugs by charge-transfer complexation of their extractable phenylhydraxones. Condensation of monosaccharides with phenylhydraxine to form the osaxones results in the introduction of basic centres, making possible charge-transfer complexation reactions with electron-accepting reagents, as described here.

*Present addresa College of Medicine and Allied Sciences, King Abdulaxix University, Jeddah, Saudi Arabia.

EXPERIMENTAL. Reagents Standard aqueous solutions (1 mg/ml) of D(+)-glucose, D( + )-mannose, D( + )-galactose, dextrose D(-)-fructose, and laevulose were prepared and diluted tenfold to give 100-pg/ml working solutions. Phenylhydraxine hydrochloride solution (50 m&ml) and IO-‘M chloroform solutions of iodine and chloranil were prepared. Preparation of osazones To 10 ml of aqueous solution containing IO-15 mg of the monosaccharide, 5 ml of phenylhydraxine hydrochloride solution were added and the mixture was heated in a glycerol bath at 110” for 10 min, cooled, transferred to a IOO-mlseparatory funnel and extracted by shaking for 1-2 mitt each time with 5 successive IO-ml portions of ethyl acetate. The extracts were all filtered through anhydrous sodium sulphate into a 50-ml standard flask and the volume made up to the mark with ethyl acetate. Colour development Procedure A. A 1.8~ml volume of the osaxone extract was transferred into a lo-ml standard flask, treated with 2 ml of iodine solution, allowed to stand for 30 min and then diluted to vohnne with chloroform. The absorbance of the resulting solution was measured in a l-cm cell at 305 mn against a reagent blank prepared in the same manner. Procedure B. A portion of the osaxone extract (equivalent to 0.3-1.4mg of the parent carbohydrate) was transferred into a IO-ml standard flask, treated with 5ml of chloranil solution and left at room temperature for 45 min or in a water-bath at 45” for 15 min, then diluted to volume with chloroform. The absorbance of the resulting solution was measured at 440 nm in a l-cm cell against a reagent blank prepared in the same manner. The concentration equivalent to parent monosaccharide in the tinal measured solution was calculated from the calibration results obtained by applying the same procedure to an appropriate range of standard solutions of the parent compound. Application to injections To a volume of sample containing about 20 mg of glucose or fructose, 5 ml of phenylhydraxine hydrochloride solution were added and the assay was completed as above.

793

MAGDA AYAD et al.

794

Determtnationof glucose in blood To a centrifuge tube containing 4 ml of isotonic solution (Na,SO,. lOH,O, 30 g/l., a 1.5-ml sample of blood or 1.0 ml of diabetic blood sample (or other body fluids to be

analysed) was transferred followed by 0. I ml of 10% sodium tungstate solution. The mixture was centrifuged for 10 min, and 1 ml of the supernatant liquid was treated with 5 ml of phenylhydraxine hydrochloride solution to form the osazone, which was then extracted with three S-ml portions of ethyl acetate. The three extracts were filtered through anhydrous sodium sulphate, and evaporated to dryness in a lO-ml standard flask. The residue was dissolved in 1 ml of ethyl acetate, and the colour was developed and the determination completed as above.

Wavetengt~

fnm)

Fig. 1. Absorption spectra of carbohydrate osazone-iodine complexes. I-Dextrose (0.093 ml/ml). II-Fructose (0.083 mg/ml).

RESULTS AND DJXUSSION

Osazone formation Phenylhydrazine

condenses with aldose and ketose to give yellow osazones.

36Onm (Figs. I and 2), which is characteristic of n-donor-iodine charge-transfer complexes.4**~~47~S

CHlOH

CHO

CH=NNHC6HI

+ CbHSNH>

I C=O

I CHOH

I C=NNHC,H,

+ NH,

I (CHOH?,

or

I (CHOW,

I 6~~0~


Ketose

Aldose

3C,HSNHN_H1

osazones are measurable spectrophotometrically but with low sensitivity. Their potential basicity initiated our study of their participation in ch~g~t~sfer complexation reactions, with the aim of increasing the sensitivity and accuracy of determination of the parent sugars. An excess of phenylhydrazine is heated with the sugar for 10 min at 110” or 20 min in a boiling water-bath, for quantitative reaction. Ethyl acetate will give complete extraction of the osazone, but then has to be dehydrated by filtration through anhydrous sodium sulphate. A blank run in the same manner has zero absorbance when measured against ethyl acetate, showing that the excess of phenylhydrazine is not extracted. A Job plot55 showed that the combining ratio of osazone (donor) and acceptor was 1: 2, as expected from the structural formula of the osazone. It is assumed that the reactive basic centre is the -NHC,H, group in view of the weak basicity of the : C = N- grouping.

-I

The

Osazone-chloranil interaction Mixing the osazone extract with a chloroform solution of chloranil resulted in development of a red colour. This is considered due to charge-transfer between the n-acceptor chloranil and the n- or x-donor osazone which, owing to the polarity of the medium, yields a coloured radical-ion complex.

C,H,

CH=N-NH-CH I C=N-

6 5 H +212=

+21,= NH-C&H5

I CzN-N:

I

' Osazonemoiety

Iodine

i6HI Outer complex

G HS

Mixing the osazone extract with iodine resulted in a change of the iodine colour from violet to yellow. This is attributed to charge-transfer between the n-donor osazone and the a-acceptor iodine, followed by the formation of an ion-association complex with t&iodide anions. The following reaction scheme is suggested. The absorption spectrum of the products has a high-intensity band with a maximum at about 305 nm and a low-intensity band with a maximum at about

C=N-N-I

I

CH=N-N-l

I

I

I I

C=N-N---I &HI Tri-iodide ion-association complex (Atnlx 305nm)

Inner complex

of monosaccharides

Determination

795

r/\,:

0.4

substitution reaction between chloranil and the secondary amino (C,H_-NH-) moiety of the osazone is excluded by the non-aqueous conditions used and the absence of the alkaline buffer necessary for such a reaction.S’*52

/M”\

/’ ’

‘1,

II

\

/’

0.2

\\

,I-\

\ ._/’

/’

A

\

,/’

Conformity to Beer’s law

01

I 260

I 340

I 300

I 360

Wavelength (nm)

Fig. 2. Absorption’ spectra of osazone&dine complexes. I-Mannose (0.096 mg/mI). II-GaIactose (0.096 mg/ml).

A linear relationship was obtained for amounts of parent monosaccharide in the range 2-12 mg. Disaccharides such as maltose and lactose gave weak and unsatisfactory reaction, indicating the poor donor activity of their osazones. Determination

From consideration of similar cases for basic drugs,49*M*S3 the following scheme is suggested.

The applicability of the procedure for determination of glucose and fructose in ampoules was

EN-$:

H-C

+ 2C~O&L,

(‘h=l in CHCL,

i C=N-rj:

I

I

&HI

Osazone moiety

-

G.O2C~.

‘3s

.lYNCC

I

&,0&L,

:N-N=C-H H

+H I.-H

H C&w

14

;N

I

C&ML,

-N=C

I GH,

I I

H : N-N=C

I I CeH,

I

Radical-ion complex =440nm) Lx

The final reaction product has an absorption maximum at 440 nm (Figs. 3 and 4), which is characteristic of the chloranil radical ion. The latter pairs with the osazone cation resulting from loss of 2 electrons per molecule of osazone, and thus modifies the wavelength of maximum absorption. The possibility of a

n

assessed with pure drugs and two commercial injections, by the standard-addition technique (Table 1). The samples were also assayed by the official method% and/or the conventional phosphomolybdate32 method (Table 2). The results in Table 1 indicate that the proposed method is fairly accurate

II c.

0.4

I’ I’

A

/’

0.2

0

//’

400

,I

\

\\ \\

‘NW_--

440

Wavelength

460

I

0.4

II ,/-\

--

A 0.2

520

.._

0

.\

/’ AI/

(nm)

‘.

/I

I 400

/

‘-__

/’

I 440

Wavelength

Fig. 3. Absorption spectra of carbohydrate osazontihloranil complexes. I-Dextrose (0.138 m&l). II-Fructose (0.131 mg/ml). TAL. ,./9-D

I 480

I 520

(nm)

Fig. 4. Absorption spectra of osaaone-chloraniI complexes. I-Mannose (0.13 me/ml). IIaalactose (0.13 mg/mI).

MAGDA AYADet al.

796

Table 1.Determination of pure glucose, pure fructose and their injections, by the proposed method and the B.P. 1953 method Recoverv,* % Official method Glucose I-test (2.3) F-test (6.39) Fructose t-test (2.3) F-test (6.39) Glucose injection (dextrose injection) t-test (2.3) F-test (6.39)

100.0 f 0.7 100.0 f 0.8 100.0 f 0.7

Iodine acceptor

Chloranil acceptor

99.7 f 0.5 1.34 1.96 99.6 f 0.6 1.49 1.77 99.8 f 0.4

99.8 f 0.4 1.12 3.06 99.9 f 0.5 0.45 2.56 99.7 f 0.4

1.49 3.06

1.60 3.06

*Mean f standard deviation of 5 determinations, calculated on nominal amount taken, sample weights 2-4 g for BP method, 15-20 mg for proposed method. The figures in parentheses are the tabulated values of t and F.

and as precise as the official method, since the calculated t and F values do not exceed the tabulated values. The proposed procedure also gave good results in the determination of blood glucose, as compared with the commonly used phosphomolybdate method (Table 3). The proposed method possesses advantages over the phosphomolybdate method in that it is more sensitive, yields a stable chromophore, gives zero blank readings, and uses simple reagents. It is suggested for use in clinical laboratories for

determining blood glucose in normal and diabetic patients. The proposed procedure for assay of glucose and fructose injections is simple, accurate, precise and more sensitive than the official methods [E.P. 1953 (Fehling titrimetry), B.P. 1980 and USP 1980 (polarimetry)], which require 2-5 g quantities of the monosaccharide. Thus, the proposed charge-transfer methods are suggested for use in pharmaceutical quality control laboratories.

Table 2. Analysis of glucose and fructose injections by the proposed method and the phosphomolybdate method Recovery,* % Phosphomolybdate method Dextrose ampoule I -test (2.3) F-test (6.39) Laevulose ampoule I -test (2.3) F-test (6.39)

100.0 f 0.5 100.2 + 0.7

Iodine acceptor

Chloranil acceptor

99.9 f 0.4 0.56 1.56 100.0 f 0.4 0.11 3.06

100.0 + 0.5 0.18 1.0 100.0 * 0.5 0.04 1.96

*Mean f standard deviation, calculated on nominal amount taken. Figures in parentheses are the tabulated values of I and F. Table 3. Determination of blood glucose by the proposed method and the phosphomolybdate method (standard-addition technique) Recovery, % Phosphomolybdate

method

99.0 99.6 99.2 101.0 100.0 *Mean f SD

I (2.3) F (6.39)

99.8 f 0.8

Iodine acceptor

Chloranil acceptor

99.5 100.0 100.0 100.0 101.0

99.5 101.0 99.6 100.0 101.0

100.0 f 0.6 0.75 1.70

100.0 f 0.8 0.55 1.0

*Mean f standard deviation; recovery calculated on standard amount of glucose added to the blood. The figures in parentheses are the tabulated values of t and F.

Determination

of monosaccharides

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