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Colorimetric Microdetermination of some Corticosteroid Drugs Using

Indophenol as Chromophoric Reagent

Article  in  Analytical Letters · July 1996

DOI: 10.1080/00032719608001502

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Alaa Sayed Amin

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Colorimetric Microdetermination of some Corticosteroid Drugs Using

Indophenol as Chromophoric Reagent
Alaa S. Amina
a
Chemistry Department, Faculty of Science, Benha University, Benha, Egypt

To cite this Article Amin, Alaa S.(1996) 'Colorimetric Microdetermination of some Corticosteroid Drugs Using Indophenol
as Chromophoric Reagent', Analytical Letters, 29: 9, 1527 — 1537
To link to this Article: DOI: 10.1080/00032719608001502
URL: http://dx.doi.org/10.1080/00032719608001502

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 ANALYTICAL LETTERS, 29(9), 1527-1537 (1996)

COLORIMETRIC MICRODETERMINATION OF SOME

CORTICOSTEROID DRUGS USING INDOPHENOL AS CHROMOPHORIC
REAGENT.

Key words : Betamethasone, dexamethasone and hydrocortisone determination,

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indophenol, spectrophotometry, dosage forms.

ALAA S. AMIN
Chemistry Department, Faculty of Science, Benha University, Benha, Egypt.

Abstract :
A simple, rapid and sensitive method for the determination of betamethasone (I),
dexamethasone (11) and hydrocortisone (III), either in the pure form or in
pharmaceutical formulations is described. The method is based on the development of
a brown product with indophenol in basic aqueous-ethanolic (50% v/v) medium. The
optimum reaction conditions for the charge transfer complex formed were assessed.
The absorbance measurements were made at 820, 816 and 822 nm for I, I1 and 111
respectively. The calibration graph was linear in the range 1-26, 1-32 and 1-35 pdml
of 1, I1 and Ill with slopes of 0.028, 0.021 and 0.024, respectively. For more
accurate analysis, Rmgbom optimum concentration ranges were 2.5-23.0, 3.0-28.5 and
3.0-33.0 @ml for I, I1 and 111, respectively. The precision of the procedure was
checked by calculating the relative standard deviation of ten replicate determinations
on a sample containing 20 pdml for each drug and was found to be 1.67, 1.39 and

1527

Copyright 0 1996 by Marcel Dekker, Inc.

 1528 AMIN

1.85% for I, I1 and 111, respectively. Many common excepience and common drugs
present in their dosage forms do not interfere, and the tolerable levels were evaluated.
Results of analysis of pure drugs and their dosage forms by the proposed method are in
good agreement with those of the British Pharmacopoeia 1993 procedure.

Introduction :
Hydrocortisone and other synthetic corticosteriods are widely used as anti-
inflammatory, antiallergenic and antireheumatic agents'. Betamethasone sodium
phosphate [ I5 1-73-51 is disodium 9a-fluoro-l I D , 17a-dihydroxy-16P-methyl-3,20-
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dioxopregna- 1,4-dien-2 1-yl orthoph-osphate, dexamethasone sodium phosphate [2392-

39-41 is a disodium 9a-fluoro- 1 1 P, 17a-dihydroxy- 16a-methyI-3,20-dioxopreg-na-
1,4-dien-2 1-yl orthophosphate and hydrocortisone acetate [50-03-31 is 1 1 p, 17a-
1 -yl acetate, were
dihydroxy-3,20-dioxopregn-4-en-2 chosen as examples of
corticosteroids for studying. Owing to their great therapeutic importance and wide
spread use, many analytical techniques have been proposed for their determination in
various samples or complicated samples. Alcoholic solutions of those compounds
which contain the primary a-ketol group on the C 1 7side-chain reduce tetrazolium salts
in the presence of tetramethylammonium hydroxide and form coloured solutions. The
absorbance of the final solution is then measured spectrophotometrically either by
allowing the reaction to proceed for a pre-selected period of time or by monitoring
the reaction rate kinetically j. High-performance liquid chromatography (HPLC) with
ultraviolet detection can be used for the determination of water soluble compounds of
these corticosteriods '.'. The official United States Pharmacopeial and British
Pharmacopoeia1 procedures are based on extraction and spectrophotomehic detection
or HPLC (I.'.

Various methods have been reported covering the determination of the studied
The aim of the present work was to demonstrate a simple, rapid and sensitive
method for determination of betamethasone (I), dexamethasone (11). and
hydrocortisone (111) either in the pure form or in pharmaceutical formulations.
 CORTICOSTEROID DRUGS 1529

ExDerimental :
Apparatus :
All the spectral measurements were recorded with a Perkin-Elmer lambda 3p
spectrophotometer, using 10-mm quartz cells. The pH measurements were made with
an Orion model 60 I Ndigital Ionalyzer.

Reagents :
All solutions were prepared from analytical-reagent grade materials using
distilled, de-ionised water.
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Stock betamethasone. dexamethasone and hydrocortisone solutions, I00 pg ml-'.

Dissolve 0.100 g of I, I I or 111 [obtained from Egyptian International Pharmaceutical
Industries Company (EIPICO)] in ethanol and dilute to IL with the same solvent. More
dilute solutions were prepared from the stock solutions by appropriate dilution with
ethanol.
Indophenol 5x10"M solution. Dissolve 0.106 g of Indophenol (Sigma product) in
absolute ethanol and dilute to 100 ml in a measuring flask with ethanol.
Phosphate buffer solutions of different pH-values (3-5-10.0) were prepared as
recommended "I.

Recommended procedure :
Transfer 0.1-3.5 ml of the sample solution into a 10-tnl measuring flask and add
1.5 ml ~ x I O - ~of
M indophenol solution and 4.5 ml of phosphate buffer solution of pH
7.4, 7.0 and 8.5 for I, II and Ill, respectively. Mix the contents well and leave for 5
min. at 25+2 "C. Dilute to the volume with ethanol and water to achieve 50% (viv)
ethanol and measure the absorbance of the brown coloured product at 820. 8 16 and
822 nm for I, I1 and 111 respectively. Use a calibration graph or empirical equation to
convert the absorbance into concentration.

Preparation of samples :
Tuhlefs : ten tablets were finely powdered. An accurately weighed portion of not
less than 100 mg was transferred into a 100-ml measuring flask and diluted to volume.
 1530 AMIN

The powder was completely disintegrated by a mechanical shaker and the solution was
filtered. From this sample solution. working solutions were prepared by appropriate
dilution, so that the final drug concentration was in the working range.
Inlec'tion so/u/ion.s : A 2.0 ml volume of the injection solution was diluted as
required so that the final drug concentration was in the working range.
Ointtnen/-creams : Weigh accurately about 1.0 g of sample and proceed as for
tablets

Results and Discussion :

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On mixing equimolar solutions of indophenol with each drug (I, I1 or 111) in basic
aqueous-ethanolic 50% (v/v) media, a new broad band in the visible spectrum at Lax
820, 816 and 822 nm for I, ll and Ill respectively was observed, [Fig. I] with
appearance of brown colour. This absorption band was ascribed to the charge transfer
complex formation, since neither indophenol nor drug alone absorb in this region.
Where indophenol alone has max at 568 nm. I has max at 241 nm, I1 at 239 nm and 111
at 240 nm in the same media.
Variable parameters affecting the complexation process were studied.When
various concentrations of the reagent were added to a fixed concentration of the drug.
1.5 ml of 5x10" M solution was found to be sufficient for the production of maximum
and reproducible color intensity. Higher concentrations of reagent did not affect the
colour intensity Different buffers (borate, acetate, phosphate and universal buffers)
were examined to reach the optimum one. Phosphate buffer solution gave the clear,
stable and high intense coloured product. The effect of pH was studied in the range 3.5
to 10.0.It was found that the maximum colour development occured at pH 7.4. 7.0 and
8.5 for I, I 1 and 111, respectively. Moreover the optimum volume of the recommended
pH value for each drug was tested and found to be 4.5 ml / 10 ml total volume, larger
amounts do not affect colour intensity.
Concerning the other optimal experimental conditions, it was found that the
complexation reaction was instantaneous at room temperature (25*2 "C). The reaction
product reached its maximum intensity through 5 minutes. The coloured product
 CORTICOSTEROID DRUGS 1531

A
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h , nm
Fig. (1) Absorption spectra of A- 7.5 x104M indophenol and A[, A11 and A111
are A + 18 pg/ml drug I, 11 and 111, respectively.

remained stable for 6hrs up to 50°C. Higher temperatures above 50 "C, lead to
dissociation of the complex formed. Many sdvmts were tested in the reaction
(methonol, ethanol, acetonitrile, acetone, dioxane and propanol). Ethanol was the best
solvent for studying due to its higher absorbance value. 50% ( v h ) ethanol was the
optimal ratio to prevent the precipitation and clear coloured solution.
On studying the molar ratio of reactants (donor : acceptor) using continuous
variation and molar ratio methods, it was found that the interaction occurs on an
equimolar basis. The reaction occurs through the formation of charge tranfer complex.
The coloured reaction prodnct can be represented, taking dexamethasone as an
example by the following structure:

0
 1532 AMIN

Table 1 : Spectral characteristics of the coloured product from the reaction between
indophenol and the studied drug 1. I1 or 111.
Parameters Drugs

Optimum pH value 7.4 7.0 8.5

Optimum A,,,,, 820 816 822
Stability constant 7.6 7.4 8.0
Beer's law limits / pdml 1-26 1-32 1-35
Ringborn range / pdml 2.5-23 3-28.5 3-33
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Molar absorptivity / I.mol-'.cm-' 1 . 4 7 ~ 1 0 ~ 1.07x10" 9.82~10~

Sandell sensitivity / pg cm-' 0.035 0.048 0.041

Quantitative parameters
Intercept - 0.009 0.013 - 0.012
Slope 0.028 0.021 0.024
Correlation coefficient 0.9996 0.9988 0.9998

Standard deviation (YO) 0.63 0.48 0.51

Range of error * (%) 1.20 1.10 1.40

Students t" (2.31)b 1.33 1.71 1.92

F-value" (2.45)' 1.87 1.99 2.16

a Comparison with the official method (7).

b Values in parenthesis are the theoretical t- and F-values for five degree of freedom
and 95% confidence limites.

The stability constants of these complexes are calculated from the data of molar
ratio and continuous variation methods applying the Harvey and Manning equation 2".

The values of the log of K, amount to 7.6, 7.4 and 8.0 for drug complexes of I, II and
111 respectively.
 CORTICOSTEROID DRUGS 1533

80 -

60 -
Tgb

40 -
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20 -

~~

0.0 0.4 0.8 1.2 1.6 2.0

log. [drug]

Fig. (2) Ringbom plots for drug I, 11 and I l l using 7.5 x loJ M indophenol

Quantifications :
A linear correlation was found between absorbance at La,for each drug and
concentration in the range given in Table 1. Correlation coefficients, intercepts and
slopes for the calibration data of drug 1, 11 and 111 are given in Table 1. Linear Beer’s
Law graphs for the investigated drugs can also be used for the calculation of
concentrations (Table I). For more accurate results, Ringbom optimum Concentration
ranges were obtained by plotting the transmittance percentage vs the logarithmic value
of concentration in pg/ml (Fig. 2). The apparent molar absorptivities, and Sandell
sensitivities of the resulting coloured products are also calculated and recorded in
Table 1.
The performance of the method was assessed by calculation of the t-and F-values
compared with the official method ’.Mean values were obtained in a student’s t- and
 1534 AMIN

Table 2 : Tolerance limits in the determination of 20 pg/ml of drugs I, 11 and I11 using
indophenol reagent.
Drug added Amount tolerated (pg/ml)
I I1 I11
Fructose. lactose. glucose > 10000 > 10000 > 10000
Galactose. maltose sucrose reserpine 8000 8500 8000
Saccharin. acetate. sodium phosphate 7000 7500 7500
Valerate. bicarbonate, magnesium stearate 5000 6000 5500
Starch, citric acid, phosphoric acid, barbiton, 3500 4000 4000
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Benzoic acid, adrenaline, acetylsalicylic acid 2000 2500 2500

Ephedrine HCI, chlorpheniramine maleate 1500 1500 1500
Chlorphenoxamine HCI, anhydrous caffeine 1200 1300 1250
Ampicillin, amoxycillin, oxacillin 1000 1100 1000
Cloxacillin. dicloxacillin, flucloxacillin 800 850 850
6.Aniinopenicillanic acid, cefadroxil, cefalexin 600 700 650
Erythromycin, neomycin sulphate 400 500 500
Norfloxacin. ofloxacin, cyprofloxcin 250 300 300
Theophylline, dyprophylline 150 175 175

F-test and 95% confidence limits for five degrees of freedom *'. and results showed
that the calculated t-and F-values did not exceed theoretical values.

Precision and Accuracy :

To examine the precision of the procedure. ten replicate analyses were performed
on the same solution containing 20 pg/inl of drug. Relative standard deviation of 1.67,
1.39 and 1.85% were obtained for drug I, II and 111, respectively.
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Table (3) : Determination of drugs I, I1 and 111 in pure and Dosage forms

Drug Sample Manufisted Labell to Proposed method Official method

content mg
Found* Recovery RSD% Found' Recovery RSD%
1 Betamethasone sodium phosphate EIPICO 5.00 4 99 99 80 0 93 5 05 101 00 137
I1 Dexamethasone sodium phosphate EPIC0 7.00 7 05 100 71 0 86 6 90 98 57 182
111 Hydrocortisone acetate EIPICO 10.00 10 05 100 50 1 13 10 20 102 00 156
I Betaderm cream EIPICO 0 lg/lOOg 0 099 99 00 0 71 0 103 103 00 2 35
I Betnovate-C cream Glaxo 0.1g/1oog 0 1006 100 60 0 54 0 103 103 00 2 50
I Diprosone cream Memphis 0.5g/ 1OOg 0 492 98 40 117 0 485 97 00 1 99
1 Betaderm ointment EIPICO 0 lg/IOOg 0 0992 99 20 0 85 0 102 102 00 173
I Diprosone ointment Memphis 0.5g/l OOg 0 494 98 80 0 I8 0 488 97 60 2 11
I1 Orazone syrup ADCO 1 Omg/l OOml 9 98 99 80 0 42 9 85 98 50 163
I1 Decadron injection EIPICO 4mg/ml 4 02 100 50 0 53 4 10 102 50 2 67
11 Dexazone tablets KPCICO 0. 5mg/Tablet 0 505 101 00 0 72 0 512 102 40 2 33
I1 Fortecortin ampoule ABICO 8mg/2ml 8 05 100 63 0 51 7 85 98 13 2 15
111 Solu-Cortef vial EIPICO 100mg/2ml 99 80 99 80 0 37 101 5 101 50 2 31
111 Nystaform ointment EIPICO ldl0Og 1 004 100 40 0 44 1022 102 20 187
I11 Neo-Cortef Drops EIPICO 15mlrjml 14.85 99.00 069 15.30 10200 2.22
a : Average of six determinations.
EIPICO : Egyption International Pharmaceutical Industries Company, Egypt.
Glaxo : Glaxo, Egypt, under licence from Glaxo Group Ltd. England.
Memphis : Memphis, Egypt, under licence from Schering Corporation, Kenilworth, New Jersey, U.S.A.
ADCO : Arab Drug Company, Cairo, Egypt.
KPCICO : Kahira Pharmaceutical & Chemical Industrial Company, Cairo, Egypt.
ABICO : Advanced Biochemical Industries Company Egypt, under Licence from E. Merck, Darmstadt, F.R. Germany.
 1536 AMIN

Intecference :
Many pharmaceutical formulations are associated with flavouring agents,
diluents, excipients such as glucose, fructose, maltose, sucrose, lactose, starch, citric
acid, benzoic acid, tartaric acid, barbitone, sodium carbonate, magnesium stearate,
reserpine, chloramphenicol, nicotinamide, and sulphaguanidine. In preliminary
experiments. these compounds were tested with indophenol reagent, and found to
interfere up to the tolerance limit listed in Table 2. Also different antibiotics,
antihistamines do not interfere. The degradation products resulted from thermal and
hydrolatic treatment also do not interfere.
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Anu!pticul upplicution :
Table 3 shows the results obtained for the determination of I. I1 and I11 drugs in
pure form and in some of their dosage forms by means of the proposed method and the
BP 1993 method ’.
The average recoveries obtained by the proposed method ranged from 98.8-
101.25. 98.9-101.1 and 98.6-101.4 YOfor the pure drugs of 1. II and 111, respectively,
and the relative standard deviations ranged from 0.86 to 1.71%, indicating high
accuracy and precision. The results obtained by the proposed procedure are compared
to those given by the BP 1993 method and thus suggest its use as an alternative to
pharmacopoeia1 method in the routine quality control of both bulk drug and
fonnulations.

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5. N.R. Scott, J. Chakraborty, and V. Marks, Anal. Biochem., 108,266, 1980.
 CORTICOSTEROID DRUGS 1537

6. United States Pharmacopeial convention, “US Pharmacopeia”, 2 I g E d n . , Mack,

Easton, PA. 1985.
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Received: December 12, 1995

Accepted: February 15, 1996

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