ACTA CHROMATOGRAPHICA, NO. 16, 2006
CHROMATOGRAPHIC BEHAVIOR
OF PERFORMANCE-ENHANCING DRUGS
ON THIN LAYERS
OF BISMUTH SILICATE ION EXCHANGER
Z. Hassankhani-Majd, V. Ghoulipour, and S. W. Husain*
Analytical Laboratory, Department of Applied Chemistry, Faculty of Chemistry,
University of Tarbiat Moallem, 49 Mofatteh Avenue, Tehran-15614, Iran
SUMMARY
The chromatographic behavior of amphetamine, bemegride, caffeine,
chlorphentermine, ephedrine, ethylamphetamine, isoproterenol, methadone,
methylendioxyamphetamine, pentazocine, pethidine, pemoline, strychnine,
and salbutamol has been studied on thin layers of bismuth silicate anion
exchanger with organic, aqueous, and mixed organic–aqueous mobile phases. Rapid separations of one drug from many other drugs and also many
quaternary, ternary, and binary separations have been achieved and are discussed. A new variable, SRF has been introduced to quantify the separating
power of the bismuth silicate anion exchanger in thin-layer chromatography.
INTRODUCTION
During the last two decades increasing use of drugs by athletes to
enhance performance has become a matter of serious concern for sports
authorities [1]. Doping analysis is regarded as an effective deterrent. The
similar chemical behavior and structures of the drugs necessitates selective
separation procedures before their estimation; gas chromatography has been
commonly used in doping control [2]. Thin-layer chromatography (TLC) is
a suitable technique for screening drugs in toxicological analysis, because
of its low cost, high speed, and easy maintenance [3,4] and the use of new
synthetic inorganic ion exchangers with high selectivity [5] has transformed
TLC into a rapid and powerful technique for separation of identical compounds [6]. We have used the ion exchanger bismuth silicate [7] with 21
mobile phases to study the TLC behavior of 14 performance-enhancing
drugs, with interesting results. Some rapid and selective methods have been
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developed for separation of one drug from others in a single-step process.
Important multiple separations of the drugs have also been achieved.
EXPERIMENTAL
Chemicals and Reagents
All chemicals and reagents were analytical grade (Merck). The drugs
studied were: amphetamine, caffeine, ephedrine, isoproterenol, methylendioxyamphetamine, methadone, pentazocine, and pethidine (Sigma), bemegride (Aldrich), pemoline, strychnine, and salbutamol (NARL), and chlorphentermine and ethylamphetamine (United Nations Office for Drug Control and Crime Prevention).
Standard Solutions
Standard stock solutions (1 mg mL−1) of the drugs were prepared by
dissolving appropriate amounts in methanol. The solutions were protected
from light and stored at 4°C.
Preparation of Ion-Exchange Plates
A solution of bismuth nitrate (0.1 M, 500 mL) in 2 M HNO3 and a
solution of sodium silicate (0.1 M in Si, 500 mL) in 2 M NaOH were mixed
dropwise with constant stirring in a flat-bottomed flask at a rate of
addition such that the contents of the flask were just neutral to methyl red
indicator. The reaction mixture was left to coagulate overnight and the gel
formed was washed five times with distilled water by decantation until the
supernatant was free from ions. The supernatant was completely removed.
A slurry prepared by mixing the gel (75 mL) with silica gel G 60 powder
(14 g), as binder, was used to coat seven 20 cm × 20 cm glass plates with
a 300 μm thick layer (Camag automatic TLC coater). The plates were dried
in an oven at 60–70°C for 2 h and then stored at room temperature inside
desiccators.
Chromatography
All glassware was acid-washed and light-resistant. Drug solutions
were applied to the plates as circular spots by means of disposable fineglass capillaries. The spots were dried completely and the plates were developed in ascending mode (without conditioning) in a Camag twin-trough
chamber. The development distance from the origin was always 12.5 cm.
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After development the plates were dried in an air oven and the drugs were
detected with the appropriate reagent (iodine vapor, of by treatment with
acid).
RESULTS AND DISCUSSION
The results recorded in Tables I and II show that rapid and selective separations of the drugs can be achieved on bismuth silicate anion-exchange plates. By using toluene–acetone–ethanol–ammonia, 4.5:4.5:0.7:0.3,
as mobile phase (Table I) isoproterenol, salbutamol, and methadone are separated from many other drugs in single-step procedures.
Table I
Separation of one drug from others on thin layers of bismuth silicate
Separation (RT–RL)*
Mobile phase
Interference
Caffeine (0.89–0.96)
from eleven other drugs
Chloroform–methanol,
9:1
Toluene–acetone–
Isoproterenol (0.00–0.02)
ethanol–ammonia,
from ten other drugs
4.5:4.5:0.7:0.3
Bemegride (0.55–0.68)
Sodium hydrogen
from ten other drugs
carbonate (0.2 M)
Toluene–acetone–
Salbutamol (0.07–0.18)
ethanol–ammonia,
from ten other drugs
4.5:4.5:0.7:0.3
Strychnine (0.10–0.18)
Methanol
from ten other drugs
Pentazocine (0.34–0.49)
from eight other drugs
Amphetamine (0.58–
0.68) from seven other
drugs
Strychnine (0.05–0.15)
from nine other drugs
Methadone (0.88–0.94)
from seven other drugs
Ethyl acetate–methanol–
acetic acid, 8:1:1
Ammonium sulfate
(0.5 M)
Methanol–water, 1:1
Toluene–acetone–
ethanol–ammonia,
4.5:4.5:0.7:0.3
Time
(min)
Bemegride, pemoline
40
Ephedrine, pemoline,
salbutamol
24
Methylendioxyamphetamine,
pemoline, caffeine
22
Ephedrine, pemoline,
isoproterenol
24
Amphetamine, isoproterenol,
caffeine
Amphetamine, chlorphentermine, caffeine, ethylamphetamine,
methylendioxyamphetamine
Ephedrine, strychnine, methylendioxyamphetamine, bemegride,
pemoline, caffeine
Amphetamine, isoproterenol,
pemoline, caffeine
Ephedrine, bemegride, pemoline,
pentazocine, salbutamol,
strychnine
36
34
25
55
24
*
RT = RF of rear of spot, RL = RF of leading front of spot
Many rapid quaternary, ternary, and binary separations of the drugs
were also achieved (Table II).
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Table II
Multiple separations achieved on bismuth silicate plates
Mobile phase
Separation (RT–RL)*
Teren (0.00–0.08)–Thidi (0.38–0.49)–Zocine (0.68–0.86)
Teren (0.00–0.08)–Thidi (0.38–0.49)–Ben (0.83–0.90)
Teren (0.00–0.08)–Thidi (0.38–0.49)–Chlo (0.82–0.90)
Teren (0.00–0.08)–Metha (0.28–0.50)–Pheta (0.61–0.86)
Teren (0.00–0.08)–Metha (0.28–0.50)–Zocine (0.68–0.86)
Teren (0.00–0.08)–Metha (0.28–0.50)–Ben (0.83–0.90)
Teren (0.00–0.08)–Metha (0.28–0.50)–Chlo (0.82–0.90)
Methanol
Teren (0.00–0.08)–Eph (0.42–0.63)–Ben (0.83–0.90)
(pure)
Teren (0.00–0.08)–Eph (0.42–0.63)–Chlo (0.82–0.90)
Strych (0.10–0.18)–Thidi (0.38–0.49)–Zocine (0.68–0.86)
Strych (0.10–0.18)–Thidi (0.38–0.49)–Ben (0.83–0.90)
Strych (0.10–0.18)–Thidi (0.38–0.49)–Chlo (0.82–0.90)
Strych (0.10–0.18)–Metha (0.28–0.50)–Zocine (0.68–0.86)
Strych (0.10–0.18)–Metha (0.28–0.50)–Chlo (0.82–0.90)
Strych (0.10–0.18)–Eph (0.42–0.63)–Zocine (0.68–0.86)
Strych (0.10–0.18)–Eph (0.42–0.63)–Ben (0.83–0.90)
Teren (0.00–0.02)–Chlo (0.46–0.50)–Thidi (0.68–0.73)–Metha (0.88–0.94)
Teren (0.00–0.02)–Chlo (0.46–0.50)–Amph (0.61–0.63)–Metha (0.88–0.94)
Teren (0.00–0.02)–Etila (0.53–0.57)–Thidi (0.68–0.73)–Metha (0.88–0.94)
Toluene–
Teren (0.00–0.02)–Etila (0.53–0.57)–Metha (0.88–0.94)
acetone–
Teren (0.00–0.02)–Chlo (0.46–0.50)–Caff (0.57–0.63)–Ben (0.70–0.77)
ethanol–
Teren (0.00–0.02)–Pheta (0.62–0.65)–Zocine (0.78–0.83)
ammonia,
Teren (0.00–0.02)–Etila (0.53–0.57)–Ben (0.70–0.77)
4.5:4.5:0.7:0.3
Teren (0.00–0.02)–Zocine (0.78–0.83)–Metha (0.88–0.94)
Teren (0.00–0.02)–Chlo (0.46–0.50)–Caff (0.57–0.63)–Metha (0.88–0.94)
Teren (0.00–0.02)–Chlo (0.46–0.50)–Zocine (0.78–0.83)
Teren (0.00–0.06)–Eph (0.57–0.62)–Pheta (0.73–0.79)–Zocine (0.92–0.96)
Teren (0.00–0.06)–Eph (0.57–0.62)–Pheta (0.73–0.79)–Thidi (0.87–0.91)
Teren (0.00–0.06)–Eph (0.57–0.62)–Pheta (0.73–0.79)–Metha (0.86–1.00)
Ethyl
Teren (0.00–0.06)–Eph (0.57–0.62)–Etila (0.77–0.81)–Zocine (0.92–0.96)
acetate–
Teren (0.00–0.06)–Eph (0.57–0.62)–Etila (0.77–0.81)–Thidi (0.87–0.91)
methanol–
Teren (0.00–0.06)–Eph (0.57–0.62)–Chlo (0.82–0.87)–Zocine (0.92–0.96)
ammonia,
Teren (0.00–0.06)–Strych (0.56–0.65)–Pheta (0.73–0.79)–Zocine (0.92–0.96)
7:12:1
Teren (0.00–0.06)–Strych (0.56–0.65)–Pheta (0.73–0.79)–Metha (0.86–1.00)
Teren (0.00–0.06)–Strych (0.56–0.65)–Pheta (0.73–0.79)–Thidi (0.87–0.91)
Teren (0.00–0.06)–Strych (0.56–0.65)–Pheta (0.73–0.79)–Zocine (0.92–0.96)
Teren (0.00–0.06)–Strych (0.56–0.65)–Etila (0.77–0.81)–Thidi (0.87–0.91)
Metha (0.02–0.16)–Chlo (0.35–0.45)–Ben (0.54–0.63)
Sodium
Zocine (0.03–0.15)–Chlo (0.35–0.45)–Ben (0.54–0.63)
hydrogen
Thidi (0.04–0.21)–Chlo (0.35–0.45)–Ben (0.54–0.63)
carbonate
Metha (0.02–0.16)–Eph (0.36–0.49)–Ben (0.54–0.63)
(0.2%)
Zocine (0.03–0.15)–Eph (0.36–0.49)–Ben (0.54–0.63)
Thidi (0.04–0.21)–Eph (0.36–0.49)
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Time
(min)
36
24
38
19
Table II (continued)
Multiple separations achieved on bismuth silicate plates
Separation (RT–RL)*
Time
(min)
Teren (0.00–0.02)–Salbu (0.20–0.26)–Strych (0.47–0.53)–Metha (0.86–0.91)
Salbu (0.20–0.26)–Thidi (0.63–0.70)–Metha (0.86–0.91)
Strych (0.47–0.53)–Thidi (0.63–0.70)–Metha (0.86–0.91)
Strych (0.47–0.53)–Zocine (0.71–0.77)
Salbu (0.20–0.26)–Strych (0.47–0.53)
Teren (0.00–0.02)–Salbu (0.20–0.26)–Strych (0.47–0.53)–Zocine (0.71–
0.77)–Metha (0.86–0.91)
24
Mobile phase
Toluene–
acetone–
ethanol–
ammonia,
20:25:3:5
*
RT = RF of rear of spot, RL = RF of leading front of spot
Amph = amphetamine, Caff = caffeine, Eph = ephedrine, Chlo = chlorphentermine, Etila =
ethylamphetamine, Teren = isoproterenol, Pem = pemoline, Pheta = methylendioxyamphetamine, Metha = methadone, Zocine = pentazocine, Thidi = pethidine, Salbu = salbutamol, Strych = strychnine.
1
0.9
0.8
0.7
0.6
Rf 0.5
0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9 10111213
substance (no.)
System 1
System 2
Fig. 1
Plots of the RF values of the drugs obtained by use of different mobile phases. System 1,
methanol–chloroform, 1:9. System 2, methanol–chloroform–ammonia, 3:5:2. 1, isoproterenol; 2, ephedrine; 3, salbutamol; 4, amphetamine; 5, methylendioxyamphetamine; 6,
chlorphentermine; 7, ethylamphetamine; 8, methadone; 9, strychnine; 10, pentazocine;
11, pethidine; 12, bemegride
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Plots of the RF values obtained for the drugs using two mobile phases are given in Fig. 1. It is clear from the pattern of the plots that slight
changes in mobile phase composition results in substantial changes in the
chromatographic behavior of the drugs, enabling the development of rapid
and selective separations.
Figure 2 shows plots of the RF values obtained for the drugs on
silica gel and on the anion exchanger bismuth silicate with toluene–acetone–ethanol–ammonia, 4.5:4.5:0.7:0.3, as mobile phase; the very different
RF values reveal the effectiveness of the anion exchanger bismuth silicate
for development of rapid and selective separations.
Rf
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9
substance (no.)
Silicagel
Bismuth silicate
Fig. 2
Plots of the RF values obtained for the drugs with toluene–acetone–ethanol–ammonia, 4.5:4.5:0.7:0.3, as mobile phase on silica gel and bismuth silicate plates.
1, isoproterenol; 2, chlorphentermine; 3, ethylamphetamine; 4, methylendioxyamphetamine; 5, amphetamine; 6, pethidine; 7, pentazocine; 8, methadone
Differences between the RF values of the drugs on bismuth silicate
layers containing a little silica gel as binder and on silica gel alone, with
methanol–chloroform, 1:9, as mobile phase are given in Fig. 3 in terms of
a new property SRF, the difference between the RF on silica gel and that on
bismuth silicate containing a little silica gel as binder, i.e.
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SRF = RF(silica gel) − RF(bismuth silicate + little silica gel as binder)
to show the effective separating power of this new adsorbent in the thinlayer chromatography of these drugs.
0.3
0.2
0.1
SRf
0
-0.1
-0.2
-0.3
0
1
2
3
4
5
6
7
8
9
10
substance (no.)
Fig. 3
Plots of the SRF values obtained for the drugs with methanol–chloroform, 1:9, as mobile
phase on silica gel and bismuth silicate plates. 1, isoproterenol; 2, ephedrine; 3, amphetamine; 4, ethylamphetamine; 5, chlorphentermine; 6, methylendioxyamphetamine; 7,
pentazocine; 8, methadone; 9, pethidine
ACKNOWLEDGMENT
The authors are grateful to Dr R. Ahmadkhaniha, Director of the
Doping-Control Laboratory, Tehran, and to Dr M.K. Rofouei, Dean of Chemistry Faculty, for providing research facilities.
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