Caffeine

CAFFEINE

l c l b h a d Uppat Zubair

khmoud M.A. Hassan,

and

Ibrahim A . AZ-Meshat

1. Description 1.1 Nomenclature 1.1.1 Chemical Names 1.1.2 Generic Names 1.2 Formulae 1.2.1 Empirical 1.2.2 S t r u c t u r a l 1.2.3 S t r u c t u r a l Confirmation by Degradation 1.2.4 CAS Registry Number 1.2.5 Wiswesser Line Notation 1.3 Molecular Weight 1 . 4 Elemental Composition 1.5 Appearance, Colour, Odor and Taste 2. Physical P r o p e r t i e s 2.1 Melting Range ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 15

71

Copyright Q 1986 by the American Pharmaceutical Association All rights of reproduction in any form reserved.

MOHAMMAD UPPAL ZUBAIR ET AL.

72 2.2

Sublimation Density 2.4 Solubility 2.5 Crystal Structure 2.6 Spectral Properties 2.6.1 Ultraviolet Spectrum 2.6.2 Infrared Spectrum 2.6.3 Nuclear Magnetic Resonance Spectra 2.6.3.1 Proton Spectrum 2.6.3.2 l 3 C - m Spectra 2.6.4 Mass Spectrum 3. Isolation of Caffeine 3.1 From Tea Leaves 3.2 From Green Coffee 3.3 Process for the Extraction of Caffeine from Coffee beans 3.4 Isolation of Natural Caffeine from the decaffeination process. 3.5 Isolation of Caffeine from Various Sources 4. Synthesis 4.1 Route 1 4.2 Route 2 4.3 Route 3 4.4 Route 4 4.5 Route 5 4.6 Route 6 5. Biosynthesis 6. Pharmacokinetics and Metabolism 7. Methods of Analysis 7.1 Elementa1 Analysis 7.2 Identification Tests 7.3 Titrimetric 7.4 Spectrophotometric 7.4.1 Colorimetric 7.4.2 Ultraviolet 7.4.3 Infrared 7.4.4 Nuclear Magnetic Resonance 7.4.5 Mass Spectrometric 7.4.6 Photo-nephlometric 7.5 Coulometric 7.6 Polarographic 7.7 Ring Oven (Micro) 7.8 Radioactive Isotopes 7.9 Radiochemical 7.10 Chromatographic Methods 7.10.1 Paper Chromatography 7.10.2 Column Chromatography 7.10.3 Thin-Layer Chrmakxagraphy 2.3

CAFFEINE

73

7.10.4 Gas Liquid Chromatography 7.10.5 High Performance Liquid Chromatography.

8.

References

9. Acknowledgements

MOHAMMAD UPPAL ZUBAIR ET AL.

74

1. Description

1.1 Nomenclature 1.1.1 Chemical Names

a) lH-Purine-2,6-dione trimethyl.

3,7-dihydro-1 3,7-

b) 1,3,7-Trimethylxanthine.

c ) Methylt heobromine

.

d) 1,3,7-Trimethyl-2,6-dioxo-l,2,3,6-tetrahydropurine.

e) 3,7-Dihydro-l,3 ,T-trimethyl-lH-purine-2,6dione. f) 1,3,7-Trimethyl-4,6-dioxopurine. 1.1.2 Generic Names

Anhydrous caffeine; Caffeine; Coffeniwn; Coffeine; Coffeina; Cuavanine; Methyltheobromine; No-Doz; Thiene. 1.2 Formulae 1.2.1 Empirical

C8H10N402 1.2.2 Structural

CAFFEINE

75 Discovered by Robiquet i n coffee i n 1821, while searching f o r quinine which he believed t o be present. I n 1827, Oudry found an a l k a l o i d i n tea and c a l l e d it t h i e n e . I n 1838, J o b s t and Mulder proved t h e i d e n t i c a l c h a r a c t e r of t h e two p r i n c i p l e s (1).

1.2.3 S t r u c t u r a l Confirmation by Degradation ( 2,3) Fischer has reported t h a t c a f f e i n e on o x i d a t i o n gave dimethylalloxane and methyl urea, i n d i c a t i n g s i m i l a r i t y with t h e skeleton s t r u c t u r e of u r i c acid.

With t h e above information it becomes evident t h a t : t h e t h i r d methyl group i s e i t h e r a t p o s i t i o n 7 o r 9 and t h e remaining oxygen atom i s e i t h e r at p o s i t i o n 6 o r 8. P o s i t i o n of t h e methyl group; Fischer a l s o i s o l a t e d another oxidation product which on hydrolysis, gave N-methylglycine, carbondioxide and ammonia. Thus, t h i s t h i r d oxidation product must be N-methylhydantoin.

co

-

C02H

+ NH3 + C02

N-methylhydantoin Therefore, it follows t h a t c a f f e i n e contains two r i n g s t r u c t u r e s t h a t of dimethylalloxane and t h a t o f methylhydantoin. The following two skeleton

MOHAMMAD UPPAL ZUBAIR E T A L .

76

s t r u c t u r e s were proposed:

F i n a l l y Fischer i s o l a t e d f o u r t h oxidation product,

N , N ' -dimethyloxamid@ CH3NHCOCONHCK3. Examination of ( I ) and (11) showed t h a t only ( I ),

could give r i s e t o t h e formation o f t h e oxamide. Therefore ( I ) must be t h e skeleton of c a f f e i n e . P o s i t i o n of oxygen atoms: t h e following two s t r u c t u r e s were p o s s i b l e with regard t o t h e p o s i t i o n of oxygen atoms.

0

a3

">

1% 0

y3

I

N/

I

CH3

I11

IV

By analogy, it would appear t h a t t h e more l i k e l y s t r u c t u r e o f I11 would be t h a t of u r i c a c i d .

CAFFEINE

77

F i s c h e r , f u r t h e r confirmed t h e c a f f e i n e s t r u c t u r e with t h e following degradation s t u d i e s : Caffeine > - *

c12

CH30H

Chlorocaffeine

NaoH

'l

Met hoxy c a f f c i n e dilute IlCl, boil

Oxycaf feine+CII Cl

3

1.2.4 CAS Registry Number

a ) Anhydrous c a f f e i n e [ 58-08-21 b ) Monohydrat e [ 5743-12-4

I

1.2.5 Wiswesser Line Notation

T 56 BN DN FN VNVJ B F H 1 . 3 Molecular Weight Anhydrous

C8HloN4O2

Monohydrate C8HloN402.H20

=

194.19

=

212.21

1 . 4 Elemental Composition C,

49.48% ; H , 5.13%

; N , 28.85% ; 0,

16.48%.

1 . 5 Appearance, Color, Odor and Taste White powder o r white, g l i s t e n i n g n e e d l e s , u s u a l l y matted t o g e t h e r . It i s o d o r l e s s and has a b i t t e r taste. 2. Physical P r o p e r t i e s 2 . 1 Melting range Between 235' and 237.5'. f o r 4 hours.

determined a f t e r drying at 80'

Hexagonal prisms by sublimation, m.p. 238?

(4)

2.2 Sublimation Caffeine sublimes a t 178? Fast sublimation i s obtained a t 160-165' under 1 mm p r e s s u r e a t 5 mm d i s t a n c e .

MOHAMMAD UPPAL ZUBAIR ET AL.

78 2.3 Density d i 8 1.23 ( 5 ) .

2.4 S o l u b i l i t y (1) 1 gm of anhydrous c a f f e i n e dissolves i n about 50 ml water, 6 ml water at 8 0 ° , 75 m l alcohol, about 25 m l alcohol a t 6 0 ° , about 6 m l chloroform, 600 ml e t h e r , 50 m l acetone, 100 ml benzene and 22 m l b o i l i n g benzene. Freely soluble i n pyrrole; i n tetrahydrofuran containing, 4% water, a l s o soluble i n e t h y l a c e t a t e and s l i g h t l y i n petroleum e t h e r .

Being a weak base, c a f f e i n e does not form s t a b l e salts, and even i t s salts of strong a c i d s , such as t h e hydrochloride o r hydrobromide, are r e a d i l y hydrolysed by water. The s o l u b i l i t y of c a f f e i n e i n water i s increased by t h e presence o f organic a c i d s o r t h e i r a l k a l i salts, e.g., benzoates, s a l i c y l a t e s , cinnamates, o r c i t r a t e s and this i s t h e reason f o r t h e use of s e v e r a l such preparations. 2.5 Crystal S t r u c t u r e Sutor ( 6 ) has determined t h e c r y s t a l s t r u c t u r e of c a f f e i n e , 1,3,7-trimethyl-2,6-dihydroxy purine (Fig. 1). Crystallographically, it is nearly isomorphous with theophylline (Fig.. 2) (7), and a comparison of bond lengths i n t h e two has yielded information concerning t h e e f f e c t s of a s u b s t i t u e n t i n t h e imidazole r i n g on t h e purine ring. The c r y s t a l s of c a f f e i n e are monoc l i n i c , space group P21/a, w i t h a = 14.8 ? 0.01, b = 16.7 ? 0.1, C = 3.97 ? 0.03 A'; 8 = 97.0 ? 0.5'. Systematic absences are hoc with h = 2n + 1, OK0 w i t h K = 2n + 1; space group ~ 2 1 / a' q h . Its f i n a l Fourier projection i s shown i n Fig. 3. Table 1 shows t h e f r a c t i o n a l coordinates r e f e r r e d t o t h e monoclinic c r y s t a l axes. The c r y s t a l s t r u c t u r e w a s solved by an application of t h e isomorphus-replacement method and a consideration of t h e possible hydrogen bond system i n t h e c r y s t a l . Bond lengths and bond angles within t h e molecule are shown i n Fig. 4, Fig. 5 and Table 2.

79

CAFFEINE

Fig. 2:

Crystal structure of theophylline.

10

15

Fig. 1:

Caffeine, showing the numbering system used.

MOHAMMAD UPPAL ZUBAIR ET AL.

80

Fig. 3:

The f i n a l UKO Fourier projection of c a f f e i n e , contours are arbitrary but equal i n t e r v a l s ; ZUO contour broken.

81

CAFFEINE

The F r a c t i o n a l C o o r d i n a t e s Referred t o t h e Monoclinic C r y s t a l Axes.

Table 1.

c2

c4 c5 '6 '8

clo c12

1' 4 N1

N3 N 7

N9 O11

1 ' 3 1 ' 5

H1 H2

H3 H4 H5 H6 H7 H8 H9 H1O

X

Y

0.2h.6 0.1003 0.0835 0.1456 0.4798 0.2879 0.1960 0.4533 0.2193 0.1796 0.ooog 0.0407

0.2225 0.2533 0.1769 0.1151 0.2481

0.0841

0.3641 0 3950

0.1418 0.2764 0.1749 0.3008

0.3057 0.1374 0.0166 0.413 0.487

0.4790

0.435 0.395 0.263

0.437 0.363 0.362

0.228

0.396 0 * 377

0.142

0.348 0.300 0.257

0.2397 0.0404

0.239 0.438

0,100

0.033 0.060

Z

0.9002 0.1295 0.1944 0.1155 0.3638 0.8790 0.9209 0.4584

X*

Y"

0.1019 0.0841 0.1463

0.2225 0.2541 0 1759 0.1143

0.4801

0.2480

0.2891

0.0832 0.3638 0.3947 0.1415 0.2769 0.1749 0.3008

0.2414

0 * 1959

0.4536 0.2196

0.9735 0.9848

0.1801

0.3376

0.0020

0.2440

0.0403 0.3063 0.1363

0.0404

0.0184

0.4705

0.7614 0.1616 0.2705 0.474 599 0.278 0.510 0

0.857 0.105 0.783 0.772 0.022

0.676

-

-

-

0.2400

-

-

x* and y* represent t h e weighted x and y coordinates from t h e hKO and hKL projections.

MOHAMMAD UPPAL ZUBAIR ET AL.

82

Fig. 4:

Bond angles and C-H bond lengths in the caffeine molecule.

10 13

Fig. 5:

14

Bond lengths in the caffeine molecules.

83

CAFFEINE

Table 2.

Bond Lengths and t h e i r Standard Deviation Bond length ( A )

Standard deviation ( A )

C2-N1

1.42

0.014

C -N 2 3

1.35

0.016

C4-N3

1.42

0.021

c4-c5

1.32

0.014

'5-'6

1.44

0.015

C6-N1

1.36

0.021

c4 -N9

1.31

0.019

'8"g

1.34

0.019

C8-N7

1.32

0.012

C -N

1.41

0.021

C1O-N1

1.48

0.016

C12-N3

1.50

0.013

C14-N7

1.47

0.018

c2-011

1.19

0.023

'6"13

1.26

0.013

Bond

5 7

A comparison is made of t h e intramolecular d i s t a n c e s with t h o s e i n o t h e r purines and a pyrimidine,Table 3, and i n d i c a t e s t h a t s t e r i c hindrance must be allowed for, i n a t h e o r e t i c a l c a l c u l a t i o n of bond l e n g t h i n r e l a t i v e l y complicated molecule of t h i s t y p e . Evidence f o r and a g a i n s t t h e e x i s t e n c e o f a s h o r t hydrogen bond between water molecules i s given.

C r y s t a l s t r u c t u r e of 2 : l molecular complexes of c a f f e i n e w i t h hexaaquamagnesium (11) bromide and hexaaquamanganese (11) T r i i o d i d e i o d i d e c r y s t a l s t r u c t u r e s of

MOHAMMAD UPPAL ZUBAIR ET AL.

a4

Table 3.

S i g n i f i c a n t l y D i f f e r e n t Bond Lengths i n a Comparison of C a f f e i n e w i t h Theophylline, Adenine Hydrochloride and U r a c i l . Caffeine with theophylline

Bond

Si g n i f ic a n t d i f f e r e n c e (A')

Actural d i f f e r e n c e (A')

c4-c5

0.04

0.05

C -N

0.05

0.07

5

7

Caffeine with u r a c i l Bond

S i g n if i ca n t difference (Ao)

Actual d i f f e r e n c e (A')

c4 -N3

0.06

0.08

c4-c5

0.05

0.09

Caffeine w i t h adenine h y d r o c h l o r i d e Bond

Significant d i f f e r e n c e (Ao)

Actual d i f f e r e n c e (A')

C2"1

0.05

0.05

C -N 2 3

0.05

0.05

C4-N3

0.06

0.06

c4-c5

0.05

0.05

C4"g

0.05

0.05

( C ~ H ~ O N ~ O ~ ) ~ M)@r2 ~ - ( Oand H~ (C~H~ON~O~)~M~(OH,)~I.I have been determined by X-ray d i f f r a c t i o n methods (83. The c r y s t a l S t r u c t u r e s O f (C8H10N402)2Mg(OH2)6Br2 ( I ) and (C8HloN402)2Mn( OH2 161. I 3 (I1) have been determined by X-ray d i f f r a c t i o n methods; c r y s t a l s o f I are tric l i n i c , s p a c e group P1, w i t h Z = 1, i n a u n i t c e l l of dimension: a = 9 . 6 2 0 ( 7 ) , b = 1 0 . 7 7 9 ( 8 ) , c = 7.645(6) Ao, a = 1 0 7 . 0 3 ( 7 ) , 8 = 1 0 8 . 8 8 ( 7 ) , y 72.71(8)O; c r y s t a l s o f

CAFFEINE

85

II are monoclinic, s p a c e group P2 / n , w i t h Z = 4 , i n a u n i t c e l l of dimensions a = 1 2 . 4 0 k ( 8 ) , b = 2 9 . 6 5 2 ( 1 2 ) , c = 9.419(6) Ao, B = 108.39(7)O. The s t r u c t u r e s of I 3nd I1 have been s o l v e d from d i f f r a c t o m e t e r d a t a by P a t t e r s o n and F o u r i e r methods and r e f i n e d by f u l l m a t r i x l e a s t - s q u a r e s t o R = 0.046 f o r I and 0.090 f o r 11. Both compounds c o n t a i n o c t a h e d r a l hexaaquametal (11) c a t i o n s , uncoordinated c a f f e i n e molecules and bromide a n i o n s i n I and t r i i o d i d e and i o d i d e a n i o n s i n 11, h e l d t o g e t h e r by a network o f hydrogen bonds. The t r i i o d i d e a n i o n s are unsymmetrical [ 1 ( 1 ) - 1 ( 2 ) = 2.89, 1 ( 2 ) - 1 ( 3 ) = 2.95A0, = 1 7 8 O ] , a r r a n g e d i n l i n e a r systems I ( 1 ) - I ( 2 ) - I ( 3) w i t h a weak ' h e a d - t o - t a i l ' i n t e r a c t i o n , t h e d i s t a n c e being o f 3.62 A O . 2.

6 Spectral Properties 2.6.1 U l t r a v i o l e t Spectrum The W spectrum of c a f f e i n e i n methanol (Fig. 6 ) was scanned from 190 t o 440 nm, u s i n g DMS 9 0 . Varian Spectrophotometer. It e x h i b i t e d a Amax at 270 nm. Other r e p o r t e d W s p e c t r a l d a t a are shown below: Solvent

2.6.2

'max

nm

E l % , 1 cm

Ref. -

Methanol

272

Trichloroethylene

278

-

Ethanol

273

5 19

11

0.1N H C 1

272

470

11

0.1N H C 1

27 5

490

12

0.1N NaOH

275

490

12

9 10

I n f r a r e d Spectrum The I R spectrum o f c a f f e i n e as KBr d i s c ( F i g . 7 ) w a s recorded on a P e r k i n E l m e r - 580 B I n f r a r e d spectrophotometer t o which an I n f r a r e d d a t a s t a t i o n is a t t a c h e d . The s t r u c t u r a l assignments

m Q)

Fig.6 UV

Spectrum

of'

CnFFeirle in /Ye!~anbl

100

2.5

4.0

3.0

wavenumber

lob0

I

2500

5.0 MICk&’5

.

2000

zo

6.0

1

f800

f60d

1

f/oO

80

9.0

10

fOm

f2

8W 1

14

s50

MOHAMMAD UPPAL ZUBAIR ET AL.

88

have been c o r r e l a t e d with t h e following frequencies Table 4 ) . Table 4 .

I R C h a r a c t e r i s t i c s of Caffeine.

Frequency cm 3110,

-1

Assignment

2950

and

CH3

1700

c=o

1650

C = N

CH s t r e t c h

Other c h a r a c t e r i s t i c bands are: 1600, 1550, 1480, 1455, 1430, 1400, 1360, 1285, 1235, 1185, 1020, 970, 860, 760 and 745. The i n f r a r e d d a t a f o r c a f f e i n e were also reported and are a s follows: 3100, 2970, 1700, 1660, 1550, 1480, 1360, 1240, 1020, 980, 750, and 610 cm-1 ( 9 ) . P r i n c i p a l peaks are 1658, 1695 and 745 cm-I (11). Major peaks 747, 1454, 1480, 1548, 1658 and 1698 cm-1 ( 1 2 ) . The region from 19-22 p i s important f o r i d e n t i f i c a t i o n of c a f f e i n e (13). 2.6.3 Nuclear Magnetic Resonance Spectra 2.6.3.1 Proton Spectrum The PMR spectrum of caffeine i n CDCl3 (Fig. 8 ) was recorded on a Varian ~ 6 0 - A , 60 MHz NMR spectrometer using TMS ( t e t r a m e t h y l s i l a n e ) a s an i n t e r n a l reference. The following s t r u c t u r a l assignments have been made (Table 5 ) . Table 5.

PMR C h a r a c t e r i s t i c of C a f f e i n e

Chemical S h i f t ( 6 ) ppm

Croup

1-N-CH3

3.53

s

3-N -CH

3.33

s

7-N-CH3

3.98

s

7.54

s

8-H ~~

s

= singlet

c

A -

I

I

3.0

_

-

.

1

.

.

.

.

l

.

2.0

.

I

.

.

I

I

.

1.0

.

.

I

11

27,87 CH3

I

The structure o f c a f f e i n e f o r carbon c h e m i c a l s h i f t s .

53

91

CAFFEINE

O t h e r r e p o r t e d d a t a are 3 . 4 , 3 . 6 , 4.0 and

7.6 ppm (7,14). Stamm(15) has i n v e s t i -

t h e a s s o c i a t i o n o f c a f f e i n e with sodium benzoate and o t h e r compounds and r e p o r t e d a l a r g e chemical s h i f t of t h e NMR s i g n a l s o f t h e t h r e e methyl Kroups. Also metal p o r p h y r i n / c a f f e i n e complexes have been i n v e s t i g a t e d ( 1 6 ) . &ed

2.6.3.2

13C-NMR

Spectra

The n a t u r a l abundance C-13 NMFf n o i s e decoupled and s i n g l e frequency o f f resonance decoupled (SFORD) s p e c t r a ( F i g s . 9 and 1 0 ) were o b t a i n e d at 20 M l z on a Varian FT-80A, 80 MHz F o u r i e r t r a n s f o r m NMR s p e c t r o m e t e r u s i n g a broad band 1 0 mm probe. The sample w a s run a t c o n c e n t r a t i o n C a 1-2 M i n d e u t r a t e d chloroform w i t h t e t r a m e t h y l s i l a n e as an i n t e r n a l r e f e r e n c e s t a n d a r d . The chemic a l s h i f t s were measured at 5 KHz s p e c t r a l width. The carbon chemical s h i f t s are a s s i g n e d on t h e basis o f t h e t h e o r y o f chemical s h i f t and SFORD s p l i t t i n g p a t t e r n and i s shown i n Table 6. Table 6. Carbon

No.

Carbon Chemical S h i f t s o f Caffeine

.

Chemical S h i f t ( 6 ) ppm

Multipli-

151.69 148.73

S

107.55

S

155.35

S

141.53

d

C-10-CH3

29.70

9

C-11-CH3

27.87

q

C-12-CH3

33.54

9

c-2

C-4

c-5 C-6 C-8

S

s = s i n g l e t ; d = doublet; q = quartet.

1000

8d 0 EbO

1

Fly.9

'3C -NMR

Norse -decoupled Spectrum oP Cp.Lf'eine in CU c/jr

93

h

MOHAMMAD UPPAL ZUBAIR ET AL.

94

2.6.4 Mass Spectrum The mass spectrum of c a f f e i n e obtained by e l e c t ron impact i o n i z a t i o n ( E I ) i s shown i n Fig. 11. It was recorded on Finigan-Mat 1020 GC/Mass spectrometer. The spectrum w a s scanned from 40 upto 440 a.m.a. Electron energy was 70 e V . The mass s p e c t r a l data are shown i n Table 7. Table 7 .

The Most Prominent Fragments of Caffeine.

m/e

Relative i n t e n s i t y

194

I loo

109

66

82

37

67

54

55

80

Fragment Base peak (M+) C5H7N3

-

Other mass s p e c t r a l d a t a were a l s o reported ( 9 $171 19411001 67" 661 , 1091661 , 821 391 42[ 281 , 40[18] and 41[16]. A comparison of mass s p e c t r a l d a t a o f theobromine, theophylline and c a f f e i n e w a s reported by S p i t e l l e r and Friedmann (18). Dunbor and Wilson have published an isotope mass spectrum method f o r i d e n t i f i c a t i o n of t h e geograp h i c a l o r i g i n of c a f f e i n e (19).

3. I s o l a t i o n of C afPeine 3.1 From Tea Leaves : Finely powdered t e a leaves (100 g ) are e x t r a c t e d with ethanol i n a soxhlet apparatus f o r 3 h r s (20). The c a f f e i n e so e x t r a c t e d i s then adsorbed on magnesium oxide. It i s t h e n desorbed after treatment with 10% H2SO4 and i s e x t r a c t e d i n t o chloroform and i s r e c r y s t a l l i s e d .

3.2 From Green Coffee : A procedure f o r t h e e x t r a c t i o n of c a f f e i n e from green coffee has been reported ( 2 1 ) . Green c o f f e e beans were steamed f o r 2 t o 3 hours t o e q u i l i b r a t e t h e water content between 40 and 50%.

100.0

1

1- 1 55

1 I

J

50.0

67

-60

100 F i g . 11.

120

140

160

EX-fiass Spectrum of C a f f o i w .

180

-1J4-

203 I -200

- - - - .222- . 1.

220

.I

- - II 240

96

MOHAMMAD UPPAL ZUBAIR ET AL. Caffeine was then e x t r a c t e d with CHC13 and c a f f e i n e upto 95% of t h e t h e o r e t i c a l amount was recovered. 3.3 A process f o r t h e e x t r a c t i o n of c a f f e i n e from c o f f e e beans by leaching w i t h water has been developed ( 2 2 ) . The highest y i e l d o f 90.4% was obtained when t h e average c o f f e e p a r t i c l e s i z e w a s 1.4095 mm, t h e water/ coffee r a t i o of 9:1, a t 7 5 O C and t h e e x t r a c t i o n was c a r r i e d out f o r t h i r t y minutes.

3 . 4 Natural c a f f e i n e is a l s o obtained as a by-product,

from t h e d e c a f f e i n a t i o n process of coffee and t e a . Some of t h e s e method a r e summarised and presented i n Table 8.

3.5 Caffeine can a l s o be obtained from various sources.

Table 9 summarises t h e source, b o t a n i c a l c h a r a c t e r i s t i c s , occurrance and t h e caffeine/theobromine content i n each case ( 3 7 ) .

CAFFEINE

97

Table 8.

I s o l a t i o n o f C a f f e i n c by Decaf f e i n a t i o n Processes.

Material

Solvent

Ref. -

Green c o f f e e beans*

F l u o r i n a t e d hydrocarbons

23

Coffee* and t e a extracts (also o i l caffeine).

With o i l s such as corn o i l , o l i v e o i l , safflower o i l .

24

Coffee base*

Benzyl a l c o h o l

25

-

26

With supercr it carbon dioxide.

27

-

28

Green coffee*

Alcohols

29

Caffeine"

S u p e r c r i t carbon dioxide with water

30

Coffee and t e a

With s u p e r c r i t carbon dioxide.

31

Roast ed co f f ee* (aqueous extracts of)

With super c ri t carbon dioxide and recovery of aroma.

32

Raw coffee*

Superc rit carbon dioxide.

33

Coffee*

Propane and butane

34

Coffee*

Aqueous ( s o l u t i o n s )

35

Coffee"

Aqueous (steam)

36

I n s t a n t coffee Black tea* Tea waste

.

Table 9.

Plant Sources of Caffeine

Caffeine

Source Seeds (beans) of

Thdobroma cacao

Information about t h e p l a n t (theobromine) con%ent ( 4 % ) Botanical c h a r a c t e r i s t i c s Native region

0,l-0.8 (0.2-2.7)

Tree, 4-15 m i n height when growing w i l d , but pruned t o 6 m under c u l t i v a t i o n . F r u i t s furrowed l e a t h e r y pods, shaped l i k e l a r g e cucumbers and cont a i n i n g 20-25 cream coloured almond-shaped seeds.

Mexico, Central and South America

West Africa

0.8-2.4

C. arabica: Shrub o r s m a l l t r e e , 4-6 m i n height when growing wild but pruned t o 2-3 m under c u l t i v a t i o n . Fruits ellipsoidal berries ( c h e r r i e s ) , r e d t o . dark purplish, containing 2 s i l v e r y skinned seeds (Beans) within t h e pulp.

E a s t Africa

South America

(stercdiaceae )

.

Seeds (beans) of

Coffea arabica, C. Ziberica, C. excelsa or C. robusta (Rubiaceae)

Main region of c u l t i v a t i o n

Continued Table 9 .

Source

Caffeine Information about t h e p l a n t (theobromine) content ( % ) Botanical c h a r a c t e r i s t i c s Native region

Seeds (beans) of

2-6

Large, climbing o r creeping p l a n t with smooth stem. F r u i t ovoid, nut-like, about as l a r g e as a grape and u s u a l l y containing 1 seed, t h e s i z e o f a hazel-nut, with white, mealy covering.

Amazon v a l l e y of Brazil

S t a t e of Amazons, Brazil.

2.7

Extensive, woody l i a n a . Stem s t o u t , up t o 12 cm i n diameter a t t h e base, with a milkywhite a s t r i n g e n t sap.

Colombia, Equador, Peru

Not c u l t i vated.

Tree, 20-30 m i n height when growing w i l d , 4-6 m under c u l t i v a t i o n . Leaves Persist e n t a l t e r n a t e , f i n e l y toothed at t h e margin and dark green i n colour.

Large a r e a i n v a l l e y s of t h e Parana, Paraguay and Upper Uruguay r i v e r s .

Paraguay and South of Brazil

Paul linia cupana

(Sapindaceae)

Bark of Pau l linia yoco (Sapindaceae)

Leaves of 1.1-1.g I l e x paraguayensis ( Aqui f o l i a c e a e )

Main region

of c u l t i v a t i o n

Continued Table 9.

Source

Caffeine Information about t h e p l a n t (theobromine) content ( I ) B o t a n i c a l c h a r a c t e r i s t i c s n a t i v e region

Leaves and shoots of

0.1-1.6

IZex vomitoria Leaves of

3-4

(Theaceae)

1.5-3.5 Seeds ( n u t s ) of Cola n i t i a k and C. acwninata (Stercdiaceae). Most o f t h e caffeine i n kolas not derived from t h e k o l a nut but added i n t h e form o f s y n t h e t i c caffeine.

of c u l t i v a t i o n

Shrub o r s m a l l tree, c l o s e l y r e l a t e d t o I. paraguayensis

E a s t c o a s t of North America from V i r g i n i a t o Mexico.

Not c u l t ivat ed .

Shrub o r s m a l l t r e e , 9-15 m i n height when growing wild, about 1.5 m under c u l t i v a t i o n . Leaves a l t e r n a t e , e l l i p t i c a l on s h o r t s t a l k s , l e a t h e r y and w i t h t o o t h e d margins.

A s s a m , China, Japan.

S r i Lanka, I n d i a , China Jaoan .

C. acwninuta: Slender t r e e , 6-9 m t a l l , t r u n k commonly

Southern Nigeria

Xest Africa

( Aqui f o l i a c e a e )

Camellia sinensis

Kain region

branching near t h e base. Bark rough and corky, grey i n colour. F o l i a g e s p a r s e , confined t o t h e t i p s o f branches. F r u i t c o n s i s t s of up t o f i v e f o l l i c l e s borne at r i g h t angles t o t h e s t a l k or s l i g h t l y downwards. F o l l i c l e s , russ e t , rough t o t h e touch. Each f o l l i c l e c o n t a i n s up t o 14 pink o r r e d seeds, covered with white s k i n . Cotyledons 3-6.

Continued Table 9.

Source

Caffeine Information about t h e p l a n t (theobromine) Native r e g i o n content ($) Botanical c h a r a c t e r i s t i c s

I n Africa, t h e seeds a r e used

+

0,

inainly as a mssticatory , but t h e y . Kay &so

be used

f o r preparat i o n of a drink (Cola).

c. nitida:

More r o b u s t t r e e ,

9-12 m h e i g h t , t r u n k unbranched for at least 1 m.

Bark smooth w i t h f i n e l o n g i t u d i n a l cracks. Foliage dense, n o t c o n f i n e d t o t h e t i p s o f t h e branches. F r u i t c o n s i s t s o f up t o f i v e f o l l i c l e s , u s u a lly bent upwards. F o l l i c l e s g r e e n , smooth t o t h e t o u c h . Seeds up t o 1 0 i n number, w i t h 2 cotyledons and of t h e shape and s i z e of horse chestnuts

.

S i e r r a Leone, Ivory coast, Ghana

Main r e g i o n of c u l t i v a t i o n

West A f r i c a , West I n d i e s South America

MOHAMMAD UPPAL ZUBAIR ET AL.

102

4 . Synthesis 4.1 Route 1 Uric acid with methyliodide gave 1,3,7-trimethyluric acid, which was converted into 8-chloroderivative and its subsequent dehalogenation afforded caffeine

(38-40).

0

0

L% H

H N

)co

N H

Uric acid

0

C'H3 1,3,7-Trimethyluric acid

CH31 NaOH

>

CAFFEINE

103

I + . 2 Route 2

C a f f e i n e h a s a l s o been s y n t h e s i s e d from uric a c i d v i a a n o t h e r r o u t e (41-42).

0

~

~

;

>

~

~

~

0

)

;

H

;

c

H

o

-~2~~~ >L20

H

N H

H

H

0

0

NHCHo

HCOM12

0

H

hYICOFHCH0

0

0

H 0

I cH3

Caffeine

MCONHCHO

MOHAMMAD UPPAL ZUBAIR ET AL.

104 4 . 3 Route 3

A d i f f e r e n t r o u t e f o r t h e p r e p a r a t i o n of c a f f e i n e from

u r i c a c i d has been r e p o r t e d ( 4 3 ) . Uric a c i d is f i r s t c o n v e r t e d i n t o 8-methylxanthine by t h e a c t i o n o f a c e t i c anhydride (44-46), a p r o c e s s which i n v o l v e s t h e i n t e r m e d i a t e formation o f a d i a c e t y l d e r i v a t i v e . The secondary amino groups are t h e n methylated i n an a l k a l i n e s o l u t i o n t o g i v e 1,3,7,8-tetramethylxanthine. F i n a l l y , t h e methyl group o r i g i n a l l y i n t r o d u c e d i n t h e 8 - p o s i t i o n i s e l i m i n a t e d ( 47 )

.

I

H

I 0 -co*

I

CH3

Caffeine

105

CAFFEINE

4 . 4 Route 4 A l k y l a t i o n o f x a n t h i n e d e r i v a t i v e s has a l s o been c a r r i e d o u t f o r t h e s y n t h e s i s of c a f f e i n e . M e t h y l a t i o n o f theobromine g i v e s c a f f e i n e (48-50), w h i l e 1methylxanthine h a s a l s o been c o n v e r t e d t o c a f f e i n e v i a t h e o p h y l l i n e (48,49,51,52). 1,7-Dimethylanalogue ( p a r a x a n t h i n e ) on a l k y l a t i o n a f f o r d s c a f f e i n e ( 5 3 ) , w h i l e x a n t h i n e on t r e a t m e n t w i t h a n e x c e s s o f methylat i n g agent g i v e s r i s e t o a ' c a f f e i n e d e r i v a t i v e

(41,49,54,55).

4.5 Route 5 C a f f e i n e i s u s u a l l y commercially s y n t h e s i z e d by T r a u b ' s methods; ( A and B, g i v e n below: ) ( 56) (A)

o=

/N H - C H 3

HOC c + %I$ 'NH-CH~ CN'

-

~

~

CH

a

NH2

1 a3

~

Z

n

/

H

2

i i . HCOOH, A

0

CH31/E t OH

0

0

0

H C

CH3

k-.--.@ i%

NaN1i2

1h3 > N

Caffeine

S

~

MOHAMMAD UPPAL ZUBAIR ET AL.

106

(B)

In this method 4-amino-5-formamido-l,3-dimethyluracil is cyclised and methylated in one step in sodium ethoxide containing methyliodide to afford initially theophylline and subsequently caffeine (57).

0

I cH3

0

CHz

L

(33

4.6 Route 6 It involves ring closure of 5-ethoxycarbonyl-1-methyl-

4-( N-methyl) ureidoimidazole, and subsequent methylation in alkaline medium gives caffeine (58).

(33 Caffeine

CAFFEINE

107

5. B i o s y n t h e s i s It h a s been r e p o r t e d ( 5 9 - 6 2 ) t h a t b i o s y n t h e s i s o f p u r i n e r i n g system probably proceeds i n n e a r l y t h e same manner i n a l l organisms as shown i n Scheme I . I n i t i a l l y , t h i s pathway was proposed f o r micro-organisms and animals b u t r e c e n t evidence s u g g e s t s t h a t it may a l s o hold good f o r h i g h e r p l a n t s . I n o s i n e monophosphate i s t h e f i r s t compound in t h i s b i o s y n t h e t i c pathway t o c o n t a i n a complete p u r i n e r i n g system (59) and it h a s a c e n t r a l p o s i t i o n i n It can be converted t o x a n t h o s i n e t h e purine m e t a b o l i s m . monophosphate i n an " I + dependent r e a c t i o n as shown below:

OH

RIBOSE

I n o s i n e monophosphate

R I BOSE-P

Xanthosine monophosphate

Xanthine, which o r i g i n a t e s from x a n t h o s i n e monophosphate by e l i m i n a t i o n o f phosphate and r i b o s e , i s t h e s t a r t i n g material f o r formation o f c a f f e i n e and o t h e r p u r i n e s . I n t h e p l a n t Coffea arabica, t h e x a n t h i n e is c o n v e r t e d , t o theobromine and t o c a f f e i n e , e i t h e r v i a N3-methylxanthine

MOHAMMAD UPPAL ZUBAIR ET AL.

108

p-o'it=? OH

OH

5-Phosphoribosyl -- pyropkosphate

-1

H

H272 ,Cao ,!j

o=c,

NH

5-Phosphoribosyl1- amine

-

I P-Ribose a-N-Formylglycinamideribonucleotide

H:'x:) P

OH

dH

P-Ribose Glycinamide ribonucleotide

H H2r"'$ NH=C,

; .

NH

ZHN

I P-Ribose

1 : )I

-

P-Ribose

N-Formylglycinamide 5-Aminoimidazoribonucleotide le-ribonucleotide

+

I

P-Ribose

I

P-Ribose

P-Ribose

5-Aminoimidazole- 5-Aminoimidazole-44-carboxylic acid N-succinocarboxamide ribonucleotide ribonucleotide

5-Aminoimidazole-4 -carboxamide ribonucleotide OH

P-Ribose 5-Formamidoimidazole-4carboxamide-ribonucleotide Scheme I.

Inosine-5-monophosphate

Biosynthesis of inosine monophosphate.

CAFFEINE

109

o r N 7-methylxanthine Scheme 11. The methyl groups o r i g i n a t e from methionine ( 6 3 - 6 5 ) .

0

I H

0

3

N -Methylxanthine

CH3

0 H

Xanthine

CHJ

\

I The0bromin e

R

CH

I

3

I

CH3 Caffeine

N 7 -Methylxanthine Scheme 11. Formation of theobromine and c a f f i n e from xanthine.

6 . Pharmacokinetics and Metabolism The absorption of c a f f e i n e from t h e g a s t r o i n t e s t i n a l t r a c t i s r a p i d b u t i r r e g u l a r (67,68). It i s d i s t r i b u t e d i n various t i s s u e s of t h e body i n approximate proportion t o t h e i r water content ( 6 3 ) . ‘The absorption o f c a f f e i n e is pH-related, an i n c r e a s e i n pH increases i t s absorption ( 6 7 ) . Within t h e t i s s u e , c a f f e i n e i s r a p d i l y broken down (69), involving metabolite r e a c t i o n s , such as N-demethylat i o n and oxidation, and r i n g cleavage ( 6 7 ) . The drug metabolising enzymes of t h e l i v e r a r e stimulated following t h e ingestion o f l a r g e amount o f c a f f e i n e ( 6 7 ) . The degree o f c a f f e i n e degradation and degradation products excreted i n t h e u r i n e o f d i f f e r e n t species seems t o vary

4;

As pa rt ic acid

\

0 C

\

N/

C 1 Fragments

Fig. 12:

Fragments

H1-CHp-C 0 0 H Glycine

Amide N of g lut amin e Origin of various atoms in the biosynthesis of caffeine.

111

CAFFEINE

considerably ( 7 0 ) . Blood l e v e l c o n c e n t r a t i o n i n four a n i m a l s p e c i e s , followinr: o r a l a d m i n i s t r a t i o n o f 25 mg/kF: [1-C1’I] c a f f e i n e , have been reported by Burger ( T O ) , (Table 1 0 ) . The plasma h a l f - l i f e f o r humans i s between 4 t o 10 hours ( 6 7 ) . Table 1 0 .

Plasma Concentration o f [1-C D i f f e r e n t A n i m a l Species.

14

] Caffeine i n

Rat

Hamster

Rabbit

Radioactivity

5.4

3.5

11.0

19.0

Caffeine

2.8

3.1

3.7

2.8

Radioac t i v i t y

0.1

0.5

0.7

0.9

Caffeine

0.1

0.1

0.7

18

18

22

Rhesus monkey

Half-life ( h )

Absorption h a l f time ( h )

Peak plasma conc e n t r a t i o n of c a f f e i n e (pg/ml).

-

13

Caffeine metabolism i n t h e rat l i v e r s l i c e s and p o s t n a t a l developing rats has been s t u d i e d (71). Rao e t al, (1973) have proposed a b i o t r a n s f o r m a t i o n r o u t e f o r c a f f e i n e i n rats (72) (Scheme 111).

MOHAMMAD UPPAL ZUBAIR ET AL.

112

>--

Hydroxylationl

Caffeine ( unchanged, 9%)

Reduct'on

N-demet hylat ions

I.

1,3,7-Trimethyldihydrouric acid (11.4%) dehydrogenation N-demethylations

I

1,3,7-Trimet hyluric acid (trace)

I

J. Theobromine

( 5.1%)

Paraxanthine (8.a%) Theophylline (1.2%)

>

3-Methyluric acid (trace)

Oxidat ion

3,6,8-Trimethylallantoin (1.3%)

I

I I Oxidat ion

I

I

V 1,6,8-Trimethylallantoic acid

I

hydrolysis

I

J.

Glyoxylic acid Met hylur ea 1,3-Dirnethylurea

Scheme 111.

Biotransformation of caffeine in rat.

113

CAFFEINE

An i n t e r e s t i n g sulphur containinE metabolite o f c a f f e i n e was i s o l a t e d from t h e u r i n e of mouse, r a t , r a b b i t and horse ( 7 3 ) , and i s shown i n Scheme I V .

0

ru

H3Nx-3 0

n

U

Caffeine

II

“i

0

CH S-CHij 2-11

/

0’

I1 Scheme I V .

N

I I11

The sulphur metabolite of c a f f e i n e .

I n case o f humans, u s u a l l y 45% o f a dose is excreted i n u r i n e i n 48 hours as 1-methylxanthine (67) and 1-methyluric a c i d (67,74). Other breakdown products excreted i n t h e u r i n e include, t h e o p h y l l i n e , lY7-dimethylxanthine, 7-methylxanthine and 1,3-dimethyluric a c i d , along with some unchanged c a f f e i n e ( 6 7 ) , Further s t u d i e s on human metabolism of (1-methyl-14C) and (2-14C)caffeine have been reported (75,76). Radiolabelled c a f f e i n e was administered ( 7 5 ) o r a l l y a t 5 mg/kg t o a d u l t , male volunteers. Blood, s a l i v a , expired C02, u r i n e , and feces were analysed f o r t o t a l r a d i o l a b e l l e d equivalents of

MOHAMMAD UPPAL ZUBAIR ET AL.

114

c a f f e i n e and i t s metabolites. High-performance l i q u i d chromatography (HPLC) was t h e main technique used f o r t h e separation of c a f f e i n e and i t s metabolites with quantitat i o n by l i q u i d - s c i n t i l l a t i o n counting. The h a l f - l i f e of c a f f e i n e i n both serum and s a l i v a was approximately 3 h r s . , with t h e amount of c a f f e i n e i n s a l i v a samples almost 65 t o 85% of t h a t found i n serum samples. The main metabolites found i n serum and s a l i v a were t h e dimethylxanthines, paraxanthine, theophyllinc and theobromine ( 7 7 ) . It has been reported (76) t h a t demethylation of t h e 3-methyl groups seems t o be t h e most important pathway i n man, and t h e plasma concentration of t h e dimethylxanthines depend on t h e i r urinary excretions a s w e l l as t h e i r own metabolism (76). Effect of c a f f e i n e on alcohol metabolism(78), basal metabolism (75) and i t s e f f e c t on exercise performance have a l s o been s t u d i e d (79). Caffeine metabolism i n sheep h a s a l s o been studied (80-82). 7. Methods o f Analysis 7 . 1 Elemental Analysis The elemental composition of c a f f e i n e i s :

Element

(83)

% Theoretical

C

49.48 %

H

5.19 %

N

28.85

0

16.48 %

7.2 I d e n t i f i c a t i o n T e s t s 1. Addition of t a n n i c a c i d s o l u t i o n t o c a f f e i n e

s o l u t i o n gives a p r e c i p i t a t e , which dissolves on f u r t h e r addition of t h e reagent ( 8 4 ) .

2. Addition of iodine s o l u t i o n and hydrochloric a c i d t o c a f f e i n e s o l u t i o n gives a brown p r e c i p i t a t e , which n e u t r a l i s e s on addition of sodium hydroc h l o r i d e (84).

3 . Reaction of c a f f e i n e w i t h potassium c h l o r a t e i n hydrochloric a c i d , and subsequent exposure t o ammonia, gives purple colour, which disappears on addition of a s o l u t i o n of a fixed a l k a l i (85).

115

CAFFEINE

4 . Addition of gold c h l o r i d e s o l u t i o n t o c a f f e i n e s o l u t i o n a f f o r d s small rods ( 8 6 ) .

5 . Addition of mercuric c h l o r i d e s o l u t i o n t o c a f f e i n e s o l u t i o n gives long needles ( 8 6 ) .

6. Comparison of t h e i n f r a r e d spectrum of t h e c a f f e i n e sample with t h a t of a reference s t a n d a r d i s a l s o employed ( 8 5 ) .

7.3 T i t r i m e t r i c Several methods have been reported f o r t h e analyses of c a f f e i n e by acid-base t i t r a t i o n procedures , e i t h e r by using d i f f e r e n t i n d i c a t o r s (87-100) o r by potentiometric methods (101-108), f o r t h e end-point d e t e c t i o n . An iodometric t i t r a t i o n method (88) w a s developed f o r

t h e determination of c a f f e i n e . It i s mixed with H2SO4 (1:l) and I B r s o l u t i o n , and t h e mixture i s d i l u t e d t o 100 m l . After f i l t r a t i o n , t o a p o r t i o n of t h e f i l t r a t e i s added K I s o l u t i o n , and t h e equivalent amount of iodine l i b e r a t e d i s t i t r a t e d with Na2S204, using s t a r c h s o l u t i o n as i n d i c a t o r . A blank is a l s o c a r r i e d out a t t h e same t i m e . Collado e t a l . , have reported a method (87) f o r t h e determination o f c a f f e i n e , phenazone, phenacetin and phenobarbitone, i n analgesic t a b l e t s . For t h e estimat i o n of c a f f e i n e , phenazone is p r e c i p i t a t e d with p i c r i c a c i d and f i l t e r e d o f f , and c a f f e i n e i s determined i o d i m e t r i c a l l y i n a c i d s o l u t i o n . Caffeine can be determined i o d i m e t r i c a l l y from icecream and cocoa admixtures (109,110). 40 g of t h e sample is heated with 1 0 m l o f water, and 1 ml of 15% aqueous NaOH, on a b o i l i n g water bath f o r 30 minutes. The mixture i s cooled and 3 m l of 30% Pb(N03)2 i s added. It i s d i l u t e d t o 100 m l with water and i s f i l t e r e d . 1 0 M l o f t h i s sample i s used t o determine c a f f e i n e t i t r imet ri c a l l y

.

The U.S. Pharmacopoeia1 method (108) c o n s i s t s i n dissolving about 400 m g of f i n e l y powdered c a f f e i n e i n 40 m l of a c e t i c anhydride. After mixing with 80 m l of benzene it i s t i t r a t e d with 0.1N p e r c h l o r i c a c i d , determining t h e end-point potentiometrically.

MOHAMMAD UPPAL ZUBAIR ET AL.

116

Caffeine has been assayed f o r another non-aqueous t i t r a t i o n procedure ( 9 3 ) . It is dissolved i n w a r m benzene and a f t e r cooling, couple o f drops of a s u i t able i n d i c a t o r , such as Sudan I V o r Nile b l u e A , are added before t i t r a t i o n with 0.1N HCL04 i n g l a c i a l a c e t i c acid. Caffeine i n cacao s h e l l s has a l s o been determined (100) by non-aqueous t i t r i m e t r i c method. About 2 g of t h e ground and de-fatted sample i s mixed with magnesium oxide and water. The mixture i s heated on a water bath f o r 30 minutes and then it i s e x t r a c t e d i n t o chloroform i n a soxhlet apparatus. The chloroform e x t r a c t is d r i e d at lO5'C f o r 25 minutes and t h e r e s i d u e is first t i t r a t e d f o r t o t a l theobromine and c a f f e i n e , and t h e n f o r theobromine only.

7.4 Spectrophotometric 7.4.1 Colorimetric A sample containing 100 mg i s dissolved i n 100 m l o f water and i s f i l t e r e d (111). A n a l i q u o t o f 5 m l i s d i l u t e d t o 25 m l and 1 ml of d i l u t e hydrochloric a c i d , and 1 m l of 10% molybdophos-

phoric a c i d are added t o it. The mixture after heating on a water b a t h i s c h i l l e d i n i c e and centrifuged. The p r e c i p i t a t e is washed and dissolved i n 25 ml o f acetone and t h e e x t i n c t i o n is measured a t 440 mu. The r e s u l t s with compounded tablets show recoveries o f c a f f e i n e , 100.2% o f t h e o r e t i c a l amount. C o r t e ' s method (112,113) c o n s i s t s i n l i b e r a t i n g c a f f e i n e w i t h concentrated H2SO4, followed by e x t r a c t i o n w i t h CHC13. It i s then estimated by a modified c o l o r i m e t r i c method involving formation o f i t s periodide ( 1 1 4 ) .

I n another method (115) c a f f e i n e (110 t o 400 pg) i s dissolved i n a f r e s h l y prepared s o l u t i o n of 0.5 m l of acetylacetone and 5 m l o f 2N NaOH. T h i s mixture i s heated at 80°C, and a f t e r cooling, a s o l u t i o n o f p-dimethylaminobenzaldehyde and 20 m l o f conc. H C 1 are added. The mixture i s f u r t h e r heated at 8OoC, and after cooling, 10 m l o f water i s added and e x t i n c t i o n of t h e r e s u l t i n g blue s o l u t i o n i s measured a t 615 mu.

117

CAFFEINE

E f f o r t s t o optimise conditions (116) have r e s u l t e d i n a n improved colorimetric method (117) f o r t h e estimation of c a f f e i n e . A small amount of c a f f e i n e is dissolved i n a mixture of 3% aqueous a c e t i c a c i d s o l u t i o n , and 10% aqueous pyridine s o l u t i o n . The mixture i s then made up t o 18 m l with water and 2 ml of N a O C l s o l u t i o n i s added. Add 2 m l of 0.1N Na2S203, followed by 3 m l of 1 N N a O H , s o l u t i o n and d i l u t e t o 50 ml with water. The e x t i n c t i o n , i n 4 cm c e l l i s measured a t 460 mp.

Iodine has a l s o been employed as a c o l o r i m e t r i c reagent f o r t h e determination of c a f f e i n e (118). 3N i o d i n e (1 ml) s o l u t i o n i s mixed with 1% s o l u t i o n of c a f f e i n e ( 5 m l ) followed by 50% H2SO4 ( 0 . 5 m l ) and i s set a s i d e f o r 1 0 minutes. The p r e c i p i t a t e i s f i l t e r e d and washed before dissolving it i n acetone. Its e x t i n c t i o n i s measured a t 525 mp. A colorimetric method f o r t h e determination of c a f f e i n e i n pharmaceutical preparations has been developed (119-121). The t e s t s o l u t i o n i s t r e a t e d with an equal volume of 10N NaOH a t 12OoC t o convert c a f f e i n e i n t o c a f f e i d i n e . It i s t h e n coupled with d i a z o t i s e d s u l p h a n i l i c a c i d and t h e e x t i n c t i o n is measured a t 451 mp. Other drugs present i n usual c a f f e i n e pharmaceutical preparat i o n s do not i n t e r f e r e .

Caffeine present i n beverages can a l s o be estimated c o l o r i m e t r i c a l l y (121-122). It i s e x t r a c t e d from t h e beverage by t h e known (123) method and is t r e a t e d with 296 s o l u t i o n o f malonic a c i d i n a c e t i c anhydride. The mixture i s heated a t 90°C, and a f t e r cooling it i s made up t o 25 m l with methanol. The e x t i n c t i o n of t h e r e s u l t i n g greenish-yellow s o l u t i o n i s measured a t 430 nm.

7. 4.2 U l t r a v i o l e t Spectrophotometric methods f o r t h e estimation of c a f f e i n e i n pharmaceutical preparations and i t s mixtures with o t h e r drugs and compounds have been developed (124-129). These i n v a r i a b l y involve

MOHAMMAD UPPAL ZUBAIR ET AL.

118

preliminary separation of c a f f e i n e , before i t s spectrophotometric estimation. An i n d i r e c t spectrophotometric method f o r t h e

determination of c a f f e i n e i n quaternary mixtures such as NaOBz, p-EtoC6H)+NHAc, aminopyrine and c a f f e i n e has been reported (1.29). The percentage of these components can be estimated by a compact c a l c u l a t i o n scheme a f t e r determination of e x t i n c t i o n c o e f f i c i e n t s a t t h e given wavelengths.

Several u l t r a v i o l e t spectrophotometric methods have been developed f o r t h e estimation of c a f f e i n e i n coffee and decoffeinated coffee ( 130-136), t e a ( 137-141) and beverages (142-146). Caffeine can a l s o be determined i n b i o l o g i c a l f l u i d s spectrophotometrically (147). O.1N-NaOH i s used t o e x t r a c t c a f f e i n e , and a mixture of s o l i d N a C l and Na2SOb is added t o enhance t h e e x t r a c t i o n o f c a f f e i n e i n t o ether-chloroform. The c a f f e i n e i s then t r a n s f e r r e d t o an aqueous a c i d i c s o l u t i o n , t h e s o l u t i o n i n t h e reference c e l l i s made strongly a c i d , and t h e contents of t h e sample c e l l are adjusted t o pH 1.3.

7.4.3 Infrared I n f r a r e d spectrophotometric method has been used (148,149) f o r t h e a n a l y s i s of caffeine. A procedure f o r t h e determination of c a f f e i n e i n pharmaceutical preparation containing aminopyrine and phenacetin has been reported (148), and t h e region 650 t o 400 cm-l o f t h e spectrum, i n KBr d i s c , w a s used f o r r a p i d and simultaneous determination of t h e t h r e e components.

7.4.4 Nuclear Magnetic Resonance A simultaneous q u a n t i t a t i v e

NMR method f o r t h e a n a l y s i s of a s p i r i n , phenacetin and c a f f e i n e i n pharmaceutical preparations has been reported (150). The method involves comparing t h e i n t e g r a l s of t h e CH2-signal ( a t 6 ppm) of piperonaldehyde, used as an i n t e r n a l standard, w i t h t h o s e of a s p i r i n methyl s i n g l e t (2.3 ppm) ,

119

CAFFEINE

phenacetin, e t h y l t r i p l e t ( a t 1 . 3 ppm) and c a f f e i n e methyl s i n g l e t ( a t 3.4 ppm). The average percent recoveries and standard deviations were 95.61 f 0.37, 96.34 k 0.47 and 101.26 -+ 1.46 f o r a s p i r i n , phenacet i n and c a f f e i n e respectively. MMR-shift technique i n forensic chemistry f o r t h e analyses of mixed samples of c a f f e i n e has been reported (151). NMR s t u d i e s o f metal porphyrin c a f f e i n e complexes (151) and associat i o n o f c a f f e i n e with sodium benzoate have a l s o been reported (152,153).

7.4.5 Mass Spectrometric A new and simple a n a l y t i c a l method using d i r e c t -

i n l e t chemical i o n i s a t ion mass spectrometry, has been reported (154) f o r simultaneous determinat i o n of c a f f e i n e and o t h e r components of a n a n t i cold drug. These components were detected as quasimolecular i o n s , and were q u a n t i t a t e d by using an accumulation program. 2

I n another method (155) B E = constant, l i n k e d scan has been used as a t o o l f o r q u a n t i f i c a t i o n s with reversed-geometry mass spectrometers. Dl a b e l l e d analogs may be used as i n t e r n a l standards, t h u s providing very simple clean-up procedures. The method i s applicable t o c a f f e i n e present i n beverages.

Caffeine w a s analysed together with o t h e r drugs l i k e a c e t y l s a l i c y l i c a c i d and phenacetin by mass spectrometry (156).

7.4.6 Photo-nephlometric Caffeine can be estimated i n t e a by using photonephlometer (157). . A c a l i b r a t i o n curve i s obtained by mixing 2 ml o f 0.01M sodium tungstophosphate, 4 m l of 2.5M HNO3 and 0.5 t o 2.5 m l of 0.001M c a f f e i n e s o l u t i o n . It i s t h e n d i l u t e d . t o 50 m l and examined i n a photo-nephlometer. Other compounds present i n tea do not cause any i n t e r f e r e n c e . With some modifications, t h i s

MOHAMMAD UPPAL ZUBAIR ET AL.

120

procedure can be used f o r t h e determination of c a f f e i n e i n presence of various d r u g s .

7.5 Coulometric Kalinowska has used coulometric technique f o r t h e of estimation of c a f f e i n e (158-162). About 0.05 c a f f e i n e i s dissolved i n hot water, and then mixed w i t h 6 m l o f 30% KOH. The mixture i s f u r t h e r heated i n a b o i l i n g water b a t h , a f t e r cooling and d i l u t i o n w i t h water, it i s n e u t r a l i s e d with hydrochloric a c i d . About 1 m l o f t h i s s o l u t i o n i s used f o r t h e determinat i o n o f c a f f e i n e with coulometrically generated c h l o r i n e , with a c u r r e n t o f 5 mA. The end-point i s determined by t h e dead-stop end method. The c o e f f i c i e n t o f v a r i a t i o n w a s about 0.5%. 7.6 Polarographic Caffeine, although not reducible a t dropping-mercury e l e c t rode can be determined polarographically a f t e r i t s oxidation w i t h bromine ( 1 6 3 ) . The wave h e i g h t s of t h e r e s u l t i n g s u b s t i t u t e d parabanic a c i d s are then evaluated. This method i s q u i t e s e n s i t i v e and t h e results obtained are within +- 3%. B a r b i t u r a t e s , a c e t y l s a l i c y l i c a c i d , ephedrine, codeine, papaverine and d i g i t o x i n do not i n t e r f e r e ; however, phenazone, phenacetin and amidopyrine should be removed b e f o r e t h e reaction. In another method (164), t h e c a f f e i n e i s analysed by anodic d i f f e r e n t i a l phase voltametry a t a glassy C e l e c t r o d e , t h e a e t e c t i o n l i m i t i s 0.5 ppm at pH 1.2. Oxidation products o f c a f f e i n e are i d e n t i f i e d by cathodic d i f f e r e n t i a l pulse polarography.

7.7 Ring-Oven

(Micro)

A r e l a t i v e l y s p e c i f i c colour r e a c t ion between chloro-

form e x t r a c t o f c a f f e i n e , a l k a l i n e acetylacetone s o l u t i o n and a c i d p-dimethyl aminobenzaldehyde solut i o n , has been used for t h e micro determination o f air-borne p a r t i c l e s of c a f f e i n e . It i s possible t o determine 0.5 pg of c a f f e i n e by t h i s method with a mean e r r o r of -+ 3% (165).

Table 11. Parameters Used f o r Paper Chromatography of Caffeine

No.

support

Developing Solvent

Detection

Ref.

1.

S & S. 2043

Consisted of s a l i c y l i c a c i d ( 0 . 3 g) dissolved i n butanol ( 30 m l ) ; t h e solut i o n being t r e a t e d dropwise with H20, t o t h e f i r s t turbidity.

Chromatogram immersed i n 1% AgNO3 d r i e d and immersed i n 0.5% aqueous K 2 C r 2 0 p .

169

2.

Whatman DE20 Anion-exchange paper.

Develop with 0.2N-aq. ammonia f o r 105 minutes.

W light

170

3.

Whatman No. 4 paper

Mobile phase w a s a xylenetetralin-n-amyl alcohol mixture and t h e s t a t i o n a r y phase w a s water a c i d i f i e d t o pH 3.2.

--

171

Paper impregnated with ethanolic s a l i c y l i c acid ( 0 . 3 g i n 30 ml) and dried.

MOHAMMAD UPPAL ZUBAIR ET AL.

122

7.8 Radioactive Isotopes Caffeine can be estimated (166) by means of isotoped i l u t i o n a n a l y s i s with [1-14C] c a f f e i n e . It i s prepared by methylation o f theobromine with [ C14]methyliodide. About 1 0 mg o f t h e p u r i f i e d sample is subjected t o combustion i n Baker's apparatus, using t h e Van Slyke-Folch reagent. The l i b e r a t e d C02 i s absorbed i n 0.25N-NaOH and 1.88% BaC12 s o l u t i o n is added. The p r e c i p i t a t e d Ba2CO3 i s f i l t e r e d o f f and i t s s p e c i f i c r a d i o a c t i v i t y i s measured with a Geiger-Muller tube. 30-80 Mg o f c a f f e i n e , even i n presence of a c e t y l s a l i c y l i c a c i d , theobromine, aminophenazone o r phenacetin give s a t i s f a c t o r y r e s u l t s . Noakes has reported (167) another radiocarbon measurement method f o r t h e estimation of natural-product p u r i t y i n case of c a f f e i n e .

7.9 Radiochemical Caffeine was a l s o analysed by using r a d i o a c t i v e t r a c e r techniques (168). It i s p r e c i p i t a t e d from t h e s o l u t i o n of t h e sample i n hydrochloric a c i d with P3*-labelled phosphomolybdic a c i d , and after f i l t r a t i o n , t h e t r a c e r I-phosphomolybdate i s dissolved i n acetone and c o l l e c t e d i n a sample g l a s s holder. The a c t i v i t y of each p r e c i p i t a t e i s determined and t h e amount calcul a t e d from a standard curve. The method i s simple and gives reproducible results with s m a l l amount of samples. 7.10 Chromatographic Methods

7.10.1 Paper Chromatoaraphy Some paper chromatographic systems used f o r t h e determination o f ' c a f f e i n e have been summarised i n Table 11. 7.10.2 Column Chromatography Various column chromatographic systems used (175-182) f o r t h e determination o f c a f f e i n e are presented i n Table 12. I n another method (182) column chromatographic technique has been employed i n i t i a l l y f o r t h e

Table 12.

Summary of Conditions Used f o r t h e Column Chromatography of Caffeine ~

support I.

Eluent

Celite 545 and 4 N &SO4 covered by a l a y e r of a mixture of c e l i t e 545 and N aq. NaHCO3.

11. Basic aluminuim oxide

111.

IV.

~~

~~

-~

Detect ion

~~

Sample

Ref.

Pharmaceuticals and c o f f e e

173

( i ) Extinction o f t h e e l u e n t i n C H C l measured a t 257, 27? and 297 m u . ( i i ) For determination i n aqueous s o l u t i o n t h e e x t i n c t i o n i s measured a t 250, 273, and 296 mu.

Coffee and i t s mixtures

174

276 mu

Beverage and tablets.

175

-

Mixture o f C H C 1 3 and diethyl ether.

Alumina (previousl y a c t i v a t e d at 800°C f o r 6 h r s . ) i n a 100 m l burett e

( i ) CHC13

( i ) Celite and ( i i )Column of c e l i t e containi n g 4N-H2S04.

( i ) Etyyl ether ( i i )CHC13

.

~~

( i i ) H20

-

Continued Table 1 2 .

V.

VI. VII.

VIII. I-

N l h

support

Eluent

Detection

Mgo-Celite 545 (l:l,w/w)

H2°

272

Celite 545

CHC13

276.5 my

Dowex 50W-X2

40% aqueous methanol

273

Multiple column containing segment s , ( a ) C e l i t e 545 ( 3 g) and 1% tartaric acid soh. (b) Celite 545 ( 3 g) and 12% H2SO4 s o l n . ( 3 ml), ( c ) C e l i t e 545 ( 2 g) and 8.4% NaHCO3 s o l n . ( 2 ml) and

Elute phenacetin with ether-CHClg (8:l) 50 m l . Dismantle t h e column e l u t e a s p i r i n from segment ( c ) and determine caffeine

(a)

Celite 545 ( 3 g) and 22.1% K3P04 soln. ( 3 m l ).

.

--

my

my

Sample

Ref.

Caffeine-containing and caffeine-free coffee

176

Coffee and decaffeinated c o f f e e

177

Pharmaceuticals

178

Pharmaceutical combin a t i o n of a s p i r i n , phenacetin, and caffeine

179

Continued Table 12.

IX.

support

Eluent

Detection

Sample

Ref.

Polyamide (0.5 g)

A f t e r passing perculate, t h e caffeine i s washed w i t h H20, 2 x 3 10 ml.

272 nm

Tea

180

H20-methanol (19:l)

254 nm

S o f t d r i n k s and food

181

x

X.

S i l i c a gel (partic l e s i z e 0.04 to 0.063 mm) i n

column (30 cm X 1 cm)

126

MOHAMMAD UPPAL ZUBAIR ET AL.

i s o l a t i o n of c a f f e i n e from a sample of decaffeinated c o f f e e , followed by i t s t i t r a t i o n w i t h 0.01M H C l O 4 using methyl v i o l e t as indicator. 7.10.3 Thin-Layer Chromatography A summary of some of t h e TLC systems i n v e s t i -

gated f o r t h e a n a l y s i s of c a f f e i n e a r e given colorimetric/speci n Table 13. Combined TLC trophotometric methods f o r t h e determination of c a f f e i n e i n pharmaceutical preparations and beverages, have been reported (183,184). The method i s based on t h e spectrophotometric estimation of t h e q u a n t i t a t i v e l y e l u t e d c a f f e i n e from TLC p l a t e s a f t e r separation from accompanying substances. TLC and high-performance TLC (1985) followed by densitometry has a l s o been employed f o r t h e determination of c a f f e i n e i n drugs ( 186,187 ) , beverages (185,188,189) and c o l a seeds (189). The s o l u t i o n from the aspirin-phenacetin-caffeine t a b l e t s i s chromatographed on Whatman chemically bonded K C 1 8 reversed-phase p l a t e s containing fluorescent phosphor and using 1:l MeOH-O.5M N a C l system (186). Caffeine w a s then determined by using a densitometer.

-

7 .lo.4 Gas Liquid Chromatography Gas l i q u i d chromatographic methods have been employed f o r t h e estimation of c a f f e i n e , and v a r i a b l e parameters used f o r some o f these a r e summarised i n Table 1 4 .

7.10.5 High Performance Liquid Chromatography High pressure l i q u i d chromatography HPLC method has wide a p p l i c a t i o n f o r t h e estimation o f c a f f e i n e i n host o f d i f f e r e n t samples. A summary o f v a r i a b l e parameters i n a few cases is given i n Table 15.

Table 13. Summary of Conditions Used f o r t h e TLC o f Caffeine -

support

Mobile phase

Detection

Sample

Ref.

Siluf ol

W 254 nm

Drugs

190

K i e s e l g e l 60F254 ( l a y e r t h i c k n e s s 0.2 mm) or C e l l d o s e Fpj4 ( l a y e r t h i c k n e s s 0.1 mm)

Spect rophotometrica l l y

Caffeine

191

Coffee

192

-

K i e s e l g e l GF 2 54

CHC13-cyclohexanea c e t i c a c i d (8:2:1)

Silica gel G

Spray w i t h 2% HgC12 s o l u t i o n containing 1 0 mg of methyl r e d p e r 100 m l . or

W 254 nm

193

Sprayed w i t h 2% HgC12 s o l u t i o n d r i e d and sprayed w i t h K I s o l u t i o n . K i e s e l g e l 60

Chloroform-acetone (9:l)

Chromatograms evalua t e d by remission dens i t o m e t r y at 273 nm w i t h a double beam

Blood

194

Continued Table 13 support

Mobile phase

Detect ion

Sample

Ref.

Cyclohexane-acet one

Determined by t h e i r quenching e f f e c t on t h e background fluorescence.

Drugs

195

Drugs

196

~

Kieselgel F

254

Silica gel G

(4:5)

--

(powders)

Table 1 4 .

Column

support

31,

Chromosorb W

OV-17 Tenax GC o r

c

Summary of Conditions Employed f o r t h e GLC o f Caffeine

5% OV17

Chromosorb WHP

SE-30

Chromosorb G

Varaport 30

Chromosorb W

3% Dexsil

300

3.5% s i l i cone SE-30

Mesh

-

Length

-

Temp. 210oc

Flow (ml/min) C a r r i e r gas

Sample

Ref.

197

Argon Flavor a n a l y s i s and beverages

198

N2(30 ml/min) (flame ionisation detector).

Drug

199

2.0m

280Oc o r N2(16 ml/min) (flame ( 310°C ionisation detector). i f sample contains papaverine)

Drug

200

1.5 glass

190°C

Drug b i o l o g i c a l material

201

0.3 m

25OoC

1.0 m

2 0 0 ~ ~

80-100

2.0 m

180Oc

-

N2(55 ml/min) H-flame i o n i s a t ion

0

(u

rl I 0

0 rl

L-

I

d

i3 m

bp

m

0 N

I

u

0

0 a3 rl

I

I

130

Table 15.

~

W,

Summary o f HPLC Conditions f o r t h e Determination o f Caffeine

Column

Mobile phase

Flow (ml/min)

Partisif 10 SCX

Aqueous s o l u t i o n of 15 mM potassium c i t r a t e , pH 3.0 and 10% (v/v) methanol

1.1 d m i n

lJ Bondapak C18, 1 0 pm

MeOH+Wat er+HOAc ( 20+79+1)

Bondapak

MeOH-O.lM, pH 7:0, citratephosphate b u f f e r (20 :80)

2a h i n

Corasil I or C o r a s i l I1

Hept ane+ethanol

Cl8 ( R a d i a l Pak A ) c a r t ridge

Wat er+MeOH+HOAc (74 :25: 1)

particle size

C18

(10:l).

Retent i o n time (m,in)

Detect i o n

Sample

Ref.

U.V.

Tea and c o f f e e

204

U.V.

Cocoa and chocolate products

205

U.V. (254 nm)

B 1ack t e a infusion

206

300 p . s . i .

U.V.

(270 nm)

Purine alkaloids

207

3.0 ml/min

U.V.

Animal d i e t s

208

-

(280 nm)

10

(280 nm)

Continued Table 15.

Mobile phase

Bondapak

0.025 M NaH2P04 i n MeOH+wat e r (2:3) t o PH 7 using NaOH

2 mUmin

Spherosil XOA 600 and XOA 800

Iso-octane+diisopropyloxide+MeOH+ t r ie thylamine+ water (34.93+49.51+ 14.58+0.20+0.78)

1d/min

Silica gel (M&N Nucleosil 50-5.5 IJM)

Methylene chloride: ethanol : water

50ml h-l

PhenylfCoras i l and C o r a s i l I1

Acetonitrile-water; 1,4 dioxan-wat er ; methanol-water; 1-4-dioxan-ac e t on i t r i l e - w a t e r and various mixtures of chloroform with methanol

C18

+,

Flow (ml/min)

Column

to 0

Retention time (min)

Detect ion

Sample

Ref.

0.48

U.V.

Drugs

209

Drugs

210

Caffeine

211

Drugs

212

(254 and

280 nm) Aprox 4 min

-

(936:47:17)

-

-

.

-

U.V.

(280 nm)

-

Continued Table l>.

Colunln

Mobile phase

Zipax SCX

O . O M HNO

--

~

0 0

5% G l a c i a l a c e t i c acid Chloroform

1.1 Bondapak

Methanol-vat er

Bondapak

c18

(30:70)

MeCN-Water

(8:92)

SpherisorbODS w i t h gradiant elution 10-pM P a r t i s i l ODS-2

Retention time (min)

4 d/min

3

Bond e l u t , C 1 8 bonded silica

c18

Flow ( d / m i n )

MeCN-H20 ( 3 0 : 7 0 )

Detection

Sample

Ref.

U.V.

Coffee

213

(254 nm)

-

-

u-Y.

Beverages

214

-

-

-

Beverages

215

1.0 d / m i n

-

Plasma

216

-

-

Beverages

217

U.V.

(273 nm)

-

-

U.V.

Decaffeinated , i n s t an t coffee and o t h e r beverages

218

-

U.V.

Appetite/suppr e s s a n t formulations

219

(272 nm)

(254 nm)

Continued Table 15. Sample

Ref.

U.V.

Drugs

220

-

U.V.

-

221

15ml/hour

U.V.

-

222

Mobile phase

Flow (ml/min)

YWGCH200x

50% s o h . of MeOH i n 0.002M K2HPO4 a d j u s t e d t o pH 8.0

1.0 ml/min

5mm

Retention time (min)

Detection

Column

(254 nm)

by H3P04. Hypersil +

x

5% Isopropanol dichloromethane

Ion 25% aqueous e t h a n o l exchange r e s i n , DowexAG 50w-x8( H+)

(280 nm) (254 nm)

Nucleosil-

MeCN-H20-HOAc ( 13:87 :1)

223

Finepak

MeOH-NH4OH ( 99 :1 and MeCN-NH40H ( 99 :1)

224

5 C18

gel-110 or Finepak SIL-CIS

Continued Table 15.

Column

Mobile phase

Bondapak C18/corasil and LI Bondapak

0.01M sod. acetate buffer, pH 5.0-MeOHt et r ahydro f u r an

Radial-Pak C 1 8 reversed phase column

Acetonitrile i n 0.1 mol/L potassium phosphate b u f f e r , pH 4.0 (9.5/90.5 by vol. )

C18

c

Flow ( d / m i n )

3 mUmin

Retention time (min)

-

Detection

Sample

Ref.

U.V.

Umbil i c a 1 cord plasma

225

Biological fluids ( neonates )

226

Various drugs sample s including caffeine

227

(254 nm)

(95:h:l)

-

.

Containing H20, acet o n i t r i l e H3PO4 and NaOH with o r without 5C8RAC ( Radial-PAK LI hexylamine Bondapak C 1 8 cartridge, HS/5C18 o r HS/3C18 o r partisil 1O-ODS- 3 Part i s i l 50DS-3RAC o r

-

-

Continued Table 15. Column

Mobile phase

Shodex

Aqueous 70% methanol at 4OoC.

D-814

(50 cm X

Flow (ml/min)

Retention time (min)

-

8 mm)

Radial-

PAK C18

0.M-phosphat e b u f f e r (pH 4) acetonitrile

Detection

p Bondapak C 1 8 (30 cm x 3.9 cm) with Bondapak C18/ Corasil guard column

-

1 0 m M-Na a c e t a t e buffer o f pH 5 methanol-t e t rahydrofuran

(95:h:l).

-

3

Ref.

270 nm Horse u r i n e and by d i f ferent i a l refr a c t ometr y

228

254 nm

Caffeine and t heophylline r eonat e s

229

U.V.

Caffeine , a s p i r i n and phenacet i n

230

254 nm

Metabolites i n Umbilical cord plasma o f bovine

2 31

(181:19).

Micro packed fused-silica, used 1 0 and 5 cm columns were used.

Sample

137

CAFFEINE

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Acknowledgements The authors would l i k e t o thank M r . Mohammad Saleem Mian of t h e Pharm. Chem. Dept., f o r h i s a s s i s t a n c e i n t h e l i t e r a t u r e review and Mr. Khalid N.K. Lodhi of t h e Central Laboratory of t h e College of Pharmacy, King Saud University f o r h i s t e c h n i c a l a s s i s t a n c e .