6.4
6.4
© Springer-Verlag Berlin Heidelberg 2005
II.6.4 Methylxanthine
derivatives
by Osamu Suzuki
Introduction
Ca eine, theophylline and theobromine are contained in co ee, chocolate (cocoa) and tea
(black and green) as weakly basic natural alkaloids.  e structures of the methylxanthine
derivatives/ xanthine derivatives including the above natural alkaloids together with synthetic
ones are shown in
> Table 4.1 [1].  eophylline, dyphylline ( diprophylline) and proxyphyl-
line are being used mainly as bronchodilators and/or heart stimulants.
Ca eine mildly stimulates the central nervous system (CNS), awakens people, relieves
them from general fatigue and activates mental activities. It has a relatively wide safe range of
doses and its estimated oral lethal dose is said to be about 10 g.  erefore, it is di cult to be
poisoned by drinking co ee or tea. Ca eine becomes problematic, when it is mixed in large
amounts with an abused drug as an adulterant; in such cases, a large amount of ca eine can be
ingested into a human body. As a fatal action of ca eine, it acts on the heart provoking supra-
⊡ Table 4.1
Structures of methylxanthine derivatives
R
1
R
2
R
3
Misc.
492 Methylxanthine derivatives
ventricular tachycardia or supraventricular arrythmia and  nally cardiac collapse to death;
especially for children, such poisoning can take place more easily [2].

GC conditions
GC column
a
: a DB-17 fused silica capillary column (15 m × 0.32 mm i. d.,  lm thickness
0.25 µm, J&W Scienti c, Folsom, CA, USA).
GC conditions
b
: an HP5890 Series II gas chromatograph (Agilent Technologies, Palo Alto,
CA, USA); detector: FID; column (oven) temperature: 140 °C → 10 °C/min → 280 °C; injection
and detector temperature: 280 °C; carrier gas: He; its  ow rate: 3 mL/min; injection: splitless
mode for 1 min followed by the split mode.
493Methylxanthine derivatives
Procedures
i. Urine specimen
i. A 5-mL volume of methanol and 5 mL distilled water are passed through a Sep-Pak C
18

cartridge to activate it.
ii. A 1-mL volume of a urine specimen, which may contain methylxanthine compound(s), is
mixed with an appropriate internal stadard (IS)
c
solution and 4 mL distilled water, and
poured into the activated cartridge with a 10-mL volume glass syringe.
iii.  e cartridge is washed with 10 mL distilled water and the target compound(s) are slowly
eluted with 4 mL of chloroform/methanol (9:1); the eluate is collected in a 4-mL volume
glass vial.
iv. A er careful removal of the upper aqueous phase, the lower organic phase is evaporated to
dryness under a stream of nitrogen.  e residue is dissolved in 100 µL methanol; 1 µL of it
is injected into GC
d

and urine, into which 10 µg each of methylxanthines had been spiked, obtained by the present
method. Although some impurity peaks appeared around 200 °C in the chromatograms ob-
tained from blank specimens, the ten methylxanthines were separated well from each other
and from impurity peaks.
494 Methylxanthine derivatives
Gas chromatograms for the extracts of human plasma and urine in the presence and absence of
ten methylxanthines using a DB-17 medium-bore capillary column. 1: caffeine; 2: theobromine
3: 1,7-dimethylxanthine; 4: theophylline; 5: proxyphylline; 6: 3-isobutyl-1-methylxanthine;
7: pentifylline; 8: dyphylline; 9: pentoxyfylline; 10: 1,3,9-trimethylxanthine. A 10-µg aliquot each
of methylxanthines was spiked into 1 mL blood plasma or urine.
⊡ Figure 4.1
495
 e recovery rates were not less than 75 % for all compounds.  eir detection limits were
0.16–0.83 µg/mL.
Except the DB-17 column, a nonpolar DB-1 capillary column had been tested; however, the
tailing phenomenon was markedly observed for theophylline, theobromine, 1,7-dimethylxan-
thine and 3-isobutyl-1-methylxanthine.  e DB-17 capillary column gave much better results
for this drug group.
> Figure 4.2 shows the metabolic pathways for ca eine.  eophylline and 1,7-dimethyl-
xanthine are the metabolic intermediates of ca eine. When ca eine is taken, the above three
compounds can appear simultaneously. It should be noted that theobromine coexists with
ca eine in co ee and tea. When both theophylline and ca eine are detected simultaneously, it
is di cult to discriminate whether only ca eine or both are taken.
Poisoning cases, and toxic and fatal concentrations
Case 1 [13]: 1-year and 2-month-old female ingested 2 g ca eine. From 1 h a er ingestion,
repeated vomitings appeared; 10 h a er, general tonic and clonic convulsion was observed. She
was subjected to emergency admission to a hospital. Upon admission, her heart beat and res-
piration rates were 170 and 40/min, respectively.  e blood ca eine concentration at 15 h a er
ingestion was 148 µg/mL. On the 2nd day of admission, adjustment of electrolytes was per-
formed, because hypopotassemia and hypochloremia appeared. On the 3rd day, the values of

a) Any capillary column with an intermediately polar 50 % phenylsilicone/50 % dimethylsili-
cone stationary phase can be used, regardless of its manufacturer.
b) Any type of GC instruments, to which a capillary column can be attached, is usable.
c) As IS, one of the methylxanthines except ca eine, theophylline, theobromine and 1,7-di-
methylxanthine listed in
> Table 4.1 can be chosen. A 10-µg aliquot of an IS is spiked into
1 mL of urine or plasma before the extraction procedure.
d) In place of GC, GC/MS can be used for analysis of methylxanthines with much higher
sensitivity and speci city, because all ten compounds dealt with in this chapter give intense
or base peaks of molecular ions in the positive EI mode [1].
e) Although blood plasma was used as a specimen in this chapter, whole blood seems usable
in place of plasma using exactly the same procedure.
References
1) Kumazawa T, Sato K, Seno H et al. (1994) Positive and negative ion mass spectrometry of ten xanthine derivatives
and their rapid clean-up with Sep-Pak C
18
cartridges from biological samples. Forensic Sci Int 68:53–67
2) Kuroiwa Y, Yoshida T (1997) Alkaloids. In: Brandenberger H, Maes RAA (eds) Analytical Toxicology for Clinical,
Forensic and Pharmaceutical Chemists. Walter de Gruyter, Berlin, pp 621–661
3) Naito H (2001) Poisoning of Industrial Products, Gases, Pesticides, Drugs, and Natural Toxins. Cases, Pathogenesis
and Its Treatment, 2nd edn. Nankodo Co., Ltd., Tokyo, pp 386–388 (in Japanese)
4) Bauza MT, Smith RV, Knutson DE (1984) Gas chromatographic determination of pentoxifylline and its major
metabolites in human breast milk. J Chromatogr 310:61–69
5) Verebey K, DePace A (1998) Rapid confirmation of enzyme-multiplied immunoassay test (EMIT) for phencycli-
dine-positive urine samples with capillary gas chromatography-nitrogen-phosphorus detection. J Chromatogr
427:151–156
497
6) Kapil R, Bruyere HJ Jr (1989) Application of capillary gas chromatography to study caffeine distribution in the
developing chicken egg. J Chromatogr 493:182–187
7) Kumazawa T, Seno H, Lee X-P et al. (1999) Extraction of methylxanthines from human body fluids by solid-