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Colorimetric Assay of Caffeine in Crude Drugs and in

Pharmaceutical Preparations
a a a
Mohamed S. Karawya , Aly Mohmed Diab & Nagla Z. Swelem
a
Department of Phermacology , National Research Centre, Dokki , Cairo, Egypt
Published online: 05 Dec 2006.

To cite this article: Mohamed S. Karawya , Aly Mohmed Diab & Nagla Z. Swelem (1984) Colorimetric Assay of Caffeine in
Crude Drugs and in Pharmaceutical Preparations, Analytical Letters, 17:1, 77-88, DOI: 10.1080/00032718408065266

To link to this article: http://dx.doi.org/10.1080/00032718408065266

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 ANALYTICAL LETTERS, 17(B1), 77-88 (1984)

Key ;;or&: Caffeine, Theobromine, Theophylline, Uur-

exide c o l o r r e e c t i o n , Coffee, Tea, Cola-
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t y p e d r i n k s , Opiate, S t r e e t samples, Det-

erminat i o n

Mohamed S. Xarawya, Aly :.iohmed Diab, and

Nagla 2. Swelem
Department of Pb,rmecology, National fiesearch Centre,
Dokki, Cairo, Egypt

ABSTRACT t
The w e l l known Llurexide r e t i c t i o n f o r purine k s e s
w a s modified and optimized for t h e q u a n t i f i c a t i o n of
caffe-e i n crude drugs, r e f r e s h i n g d r i n k s and pharma-
ceuticals. The method w a s a l s o a p p l i e d f o r t h e detec-
t i o n and a s s a y o f c a f f e i n e i n mixtures of i l l e g a l opi-
ate samples. It waa a l s o a p p l i o d t o a s s a y theobrombe
a n d t h e o p h y l l i n e a l o n g s i d e w i t h caffeine i n their nat-
u r a l and synt-tic m i x t u r e s a f t e r a simple e e p a r a t i a g
procedure depending on their d i f f e r e n t b a a i c i t i a s .

77

Copyright 0 1984 by Marce.1 Dekkcr, Inc. 0003-27 19/84/1701 OO77$350/0

 7a KARAWYA, DIAB, AND SWELEM

IYL’mD1JCTIOE :

CaYeine i s one of t h e most widely ingested natural

alkaloids and i n some countries the p e r c a p i t a annual
consumption may be a s high 85 100 g. There has been an
Utmost i n t e r e s t in t h e c l i n i c a l , toxicological and
t e r r a t o g e n i c e f f e c t s of chronic c a f f e i n e consumpt ion.
U n t i l recently, t h e usual technique f o r estimation of
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caffeine i n r a w material, pharmaceuticals or biological

f l u i d s has been UV-spectrophotoncitry, which proved t o
be n e i t h e r s a t i s f a c t o r y nor specific. O t h e r m e t h o d s i n c l u d e
colorimetry a n d G L C o n HPLC”*. A radioimmuno-

assay procedure which is highly s p e c i f i c has been

described for c a f f e i n e i n plasma and s a l i v a but r e q u i r e s
expertise i n t h e preparation of c a f f e i n e antibody. In
S8arChing f o r a simple and s p e c i f i c assey f o r c a f f e i n e
4
t h e well known h r e x i d e q u a l i t a t i v e color r e a c t i o n
was considered as a nice start. As no trials t o quantify
t h e r e a c t i o n were t r a c t a b l e i n t h e l i t e r a t u r e , it i s
intended i n t h i s work t o study i t s experimental condi-
t i o n s t o suit t h e q u a n t i t a t i v e assay of c a f f e i n e i n raw
material and i n pharmaceuticals.

MEZHOD :
Estimation of Caffeine i p Standards:
Weight accurately 0.1 g caffeine, tranaf er quant lvely

t o a 100 - ml measuring f l a s k , dissolve i n ethanol and

adjust t o volume with the s&me solvent. P i p e t t e 0.1 ml
 COLORIMETRIC ASSAY OF CAFFEINE 79

of t h e s o l u t i o n ( = 100 p g caffeine) i n t o a procelain

dish, add 0.1 g K B N 3 and 1 rdL H C 1 and evaporate t o
dryness on a water bath. Leave t o cool, then dissolve
t h e residue i n 5 ml dimethylformamide, leave t o stand
f o r 5 minutes and measure t h e developed reddish brown
c o l o r at 500 LIPL against a reagent blank. A calibra-
t i o n curve of 20 - 120 r@;/a
E.color i n t e n s i t y w a s
found t o obey Beer's l a w w i t h a standard deviation of
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-+ 1-25.
Estimation of Caffeine i n Coffee and Tea :

Transfer 10 g of powdered coffee or t e a t o a 500 ml

beaker. Add 100 ml of water and b o i l the mixture gently
f o r 15 min, with continuous s t i r r i n g . S t r a i n t h e hot
e x t r a c t through m s l i n , e x t r u d i n g as much l i q u i d 88 possible.
Wash the marc on the muslin w i t h 100 m l of b o i l i n g water
a d e x t r u d e again. Add a s o l u t i o n o f 10 %
! basic lead
a c e t a t e t o the f i l t r a t e u n t i l na more p r e c i p t a t e w a s for-
md , Heat t h e mixture t o b o i l i n g and f i l t e r rapidly
t h r o u g h a Buchner funnel. Wash t h e residue on the f i l t e r
with t w o successive 40 m l portions of boiling water ,
heat t h e f i l t r a t e again to b o i l i n g and add d i l u t e H2S4
u n t i l no more p r e c i p i t a t i o n occurs. Add one g of
decolorizing cnarcoal t o t h e mixture, boil for a f e w
minutes and f i l t e r . Collect the f i l t r a t e i n a large
evaporating diah. Evaporate t h e f i l t r a t e t o 50-70 ml
over a small flame and leave t o cool. Transfer t h e
concentrate t o a Beparatmy funnel, make alkaline
 80 KARAWYA, DIAE, AND SWELEX

dth H d H or m40H and extract w i t h CHC13 (" I 10 a).

Evaporate the co*ined chloroform extract and dissolve
the residue i n 10 pl ethanol i n a volwmtric flaak.One
ml of the resulting aolution w a pipetted
~ in an evap.
orating dish and evaporated t o dryness and complete the
aseey as under caffeine standards commencing from the

mrds " add 0.1 g KBr03" .

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Eetilaation of Gaffeine in BefreehinR Drinks x

Accurately measure 50 ml of a degaased caca-cola

d r i n k , render akaline with dilute N d H (5 ml) and extract
with CHC13 ( 3 x 20 nil) . F i l t e r the combined CHC13 extract,
rmh w i t h dilute NaOH (5niL) and 0.02 YL K h 0 4 (20 a),
evmorate t o drynee6 and complete the assay as described
before.

Estimation of Caffeine i n Pharmaceutical Preparationex

l-conpoun d Caffeine Tabletsr

Product I : (each tablet contains : caffeine 20 pg,

chlorpheniramine malate).

Product 11: (contains t caffeine 25 mg, Paracetamol,

codeine phosphate end phenobarbitone)

Product IIIt(containa x c d f e i n e 60 ag, ergotapline

tart arat e)

Accurately r s i g h a quantity of finely p o e t & .

 COLORIMETRIC ASSAY OF CAFFEINE 81

20 tablet8 equivalent t o rC0 mg caffeine, triturate

with 20 ml 10 % HC1, transfer t o a separator and
extract w i t h CHC13 (4 x 20 ml). Evaporate the combined
chloroforn extract t o dryness c n a water bath and
complete the assqy as before,

240m~oundCaffeine Suppositarig
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(each mpp- containe caffeine 55 rmg, ergotamhe

tartarate, anneurine nitrate, ethoqybenzamide). Dissolve
10 supp. in h o t 10 96 HC1, add 2 g PJaCl and extract t h e
mixture w i t h ether (3 I 40 ml) . Re-extract the remaining
acid solution with chloroform (4 x 20 isl) . Evaporate
the C;HClJ extract and complete Ithe assay as Blentioned
before.

Application of Modified Ihrexide Method for the h a w of

Caffeine i n SiBplated IllegalmSt;reetnOpiate Samples :

For the clandestine nwnfttcture of heroin fmm

opium, the l i m e w t h o d has betcorn the procedure m8t
often used i n the preparation of raw material for this
mwwse , becaarse of ita S i B p l i C i t y and th e pinipal
amorUrt Of Cheraicals required.

Crude heroin so-prepared was found t o contain

other minor conetituemts of remarkable forensic value
v i a . mnoacetylmrphine and acet,ylcodedne. Other minor
 82 KARAWYA, D I A B , AND SWELEM

c o n s t i t u e n t s derived from opium are thebaine, narcotine

etc.... . This mixture i s being subject t o adult-
e r a t i o n and d i l u t i o n processes. Caffeine is reported
t o be a W o r diluent in most of heroin eiezures 5 .
Procedure : To t e s t t h e proposed colorimetric method
f o r t h e estimation of c a f f e i n e in such complex i l l e g a l
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opiate samples, 5 s y n t h e t i c mixtures sipnlating such

samples were prepared ; each mixture contained appro-
ximately (%): morphine 10, codeine 2 , acetyl morphine
0.05 , heroin 0.01 , acetyl codeine 0.05 , narcotine
0.01 and thebaine 0.01.
5, 10, 20 30 , 40 mg of c a f f e i n e reference powder
(accurately weighed) were added t o t h e 1& ,2
d , 3& ,
n
4 2 and 5% mixture respectively. Each mixture w a s die-
solved i n 20 ml 10 % H C l and e x t r a c t e d with CHC13(3z10ml).
The chloroform e x t r a c t w a s washed with 5 m l of H20 and
t h e washings were rejected. Chloroform w a s evaporated
on a w a t e r bath and the residue w a s dissolved i n 10 m l
ethanol. An aliquot of 0.2 ml of each of t h e 5 ethanolic
solutions were p i p e t t e d into 5 separate porecalain
dishes, and t h e modified k r e x i d e color method was corn-
p l e t e d as described before.

BESULTS AM) DISCUSSIOA :

Being highly s p e c i f i c for purine baees, quantifica-

t i o n of Idurexide reactiol? w a s the goal of s e v e r a l workers,
 COLORIMETRIC ASSAY OF CAFFEINE 83

but i n vain. In this work, however, fortunately enough, the

r e a c t i o n could be modified and omployed far the first t i m e
t o assess caffeine, theophylline end theobrolaine. The
o r i g i n a l k s e x i d e r e a c t i o n depends upon t h e oxidative
cleavage (using KC103 and HC1 or BNo3) of c a f f e i n e ( or
o t h e r purine bases) i n t o an alloxantin derivative which
rearranges i n t o amalic acid ; t h e l a t t e r on exposure t o
ammonia acquires a v i o l e t color.
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In t h e present work the s t e p of exposure t o emmonia

was omitted and t h e residue w a s dissolved d i r e c t l y i n

dimethylformamide where a reddish brown color a e m

able at 500 Dp1 appeared. This modification w a ~found
necessary 85 n e i t h e r the amount of ammonia vapours nor
t h e time of exposure were easy t o quantify. Another
modification which furnished higher s e n s i t i v i t y of t h o
nethod was t h e use of KBr03-HC1 inetead of KC103-HC1 ;
w i t h t h e l a t t e r low r e s u l t s were obtained. In dimetbJllf0-
raami.de t h e r e a c t i o n product (amelic acid) undergoes par=
t i a l i o n i z a t i o n giving reddish brown color. Other n a t u r a l
xanthine bases (theobrodne) and theophylline) as w e l l as
s y n t h e t i c xanthine derivatives ( 18. €5. PropoKYp~lline)
r e a c t similarly yielding f i n a l l y analic acid derivatives.
Therefore, these substances, i f w e s e n t , should be removed
from t h e sphere of r e a c t i o n e.g. by !FLC.

In coffee and tea , caffefine could be separated

from theophylline and theobromin13 by v i r t u e of its
 relatively much weaker basicity enabling it t o be
cted by chloroform fran ammonia o r dilute sodium hydro-
xide alkaline mixtures. Thin-layer chromato-
graphic patterns of t h e three xanthine bwes before and
after alkalixisation e t h ammonia followed by extraction
with chloroform Proved t h i s behavior. Dilute sodium
hydroxide solution was reported t o play the same
r o l e as hydroxide, especially in high con=
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epratonium

centrations of theobromine. Ammonia, quaternary amtuoniurp

hydroxides and a l k a l i metal hydroddes dissolved the
reaction products readily giving colored salts 8 but
the color faded rapidly. Water also gave an unetable
intense violet color vihich faded steadily. D i m e t b y l -
fornamide proved t o be t h e best medium i n e i c h a rel-
atively stable reddish brown color w a s obtained.

Crude drugs (coffe and tea) , soft drinks (cola-

type drinks) a d various dosage form8 (tablete, suppo-

s i t o r i e s ) were analyeed f o r caffeine content with the
proposed colorimetric reaction (Table 1, 2 )*

Moreover, caffeine is known t o be a common diluent

i n i l l e g a l opiate preparation Motives for dilu-
t3.q such preparations d t h coffeine are t

1- The cheapness and eaey availability of caffeine in

relation t o the highly and ever increasing prices

of the a a i n narcotic constituents of these prepara-
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T a b l e (1)

Doteraination o f caffeine In pparmacentical €beparetiom by the Proposed and U d 4 ) methods

Stated Found Found 96. Recav. ’

PFeparation vr Added
‘8nrexlde kra. Bur. uv Rur. W
Tablet: I 20 20 1905 19.8 20 39 3905 97.5

Qafgeine supp- 55 55 53.9 54 55 107.9 108 94.5

. ,.-. I
- . . . .,. . .. . ..- - ., . . - .. ..
 86 KARAWYA, DIAB, AND SWELEM

Table 2

Assay of caffein i n coffee, t e a and coca c o l a by modified

k r e x i d e method

Itcn Percentage of Caffein

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Tea (yellow l a b e l )

Tea El-hmhria

Coca- c o l a

t ions ( e. g. morphine, heroin.. .... e t c).

2- The b e l i e f of dealers t h a t c a f f e i n e (among o t h e r

d i l u e n t s and a d u l t e r a n t s ) a c c e l e r a t e s the onset o f
narcotic a c t i o n of these preparations and elongates
i t s duration.

Assay o f c a f f e i n and other a d u l t e r a n t s i n such

illegal samples i s very important as it may help t o

t r a c e the r o u t e t h a t the sample followed i n its way t o
t h e u s e r through successive dealers. The proposed
colorimetric reaction, although highly s p e c i f i c and
 COLORIMETRIC ASSAY OF CAFFEINE a7

Table 3
Determination o f c a f f e i n i n simul-ated Illegal o p i a t e samples
with modified Ihrexide Method

Mixture add found % recov.

I 5 4.82 96.6
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I1 10 9-7 97
I11 20 19.25 96. 25

IV 30 29.1 97.

V 40 39.2 98

Average recovery = 96.97 %

s e l e c t i v e f o r caffeine necessita.tes i n most cases t h e

separation of caffeine from such complex mixtures aa
food, drinks or siezures, p r i o r t o applying the color
reaction.

The r e s u l t s obtained from crude drugs, pharmaceuti-

c a l s , refreshing drinks an0 i l l e g a l opiate samples (Table
1, 2 , 3 ) revealed the efficiency of recovery i n compari-
son d t h t h e uv spectrophotometric method.
 88 KARAWYA, DIM., AND SWELEM

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Received October 25, 7983

Accepted November 18, 1983