Short Report Ann Clin Biochem 2001; 38: 64±66

Evaluation of thin-layer chromatography methods for laxative

detection
A Duncan1 and I J Phillips2
From the 1Department of Clinical Biochemistry, Macewan Building, Royal In®rmary, North
Glasgow University Hospital NHS Trust, Glasgow G31 2ER, Scotland, and the 2Department
of Chemical Pathology (NHS), Level D, University Hospitals NHS Trust, Southampton
SO16 6YD, UK

INTRODUCTION each stage in methodology, several identical

Surreptitious laxative abuse is a signi®cant cause
of chronic diarrhoea, the detection of which is
often missed, leading to misdiagnosis and
additional costly investigations. Affected pa-
tients usually deny taking laxatives, and so the
diagnosis can only be made by a search for
laxatives among the patient’s belongings or by
chemical analysis of urine. Ethical and practical
considerations make the latter approach prefer-
able. A pilot National External Quality Assur-
ance Scheme (NEQAS) for laxatives has
revealed poor analytical performance, with
overall false negative and false positive rates of
28% and 10%, respectively.1 In an attempt to
optimize laxative detection we have individually
evaluated the extraction, chromatography and
detection stages of thin-layer chromatography
(TLC) methods described in the literature or in
current use by NEQAS participants.
MATERIALS AND METHODS
Ten urine samples, four negative and six positive
for laxatives (three for aloe-emodin, two for
rhein, two for emodin, two for phenolphthalein
and two for bisacodyl metabolite), were ob-
tained from the pilot NEQAS scheme. The
samples were deconjugated overnight at room
temperature using 30 mL glucuronidase2
(132 500 units/mL, G7017 from Sigma Chemical
Co., Poole, Dorset). Standard solutions (2 mg/
10 mL in chloroform) were prepared for rhein,
aloe-emodin,emodin, phenolphthalein,danthron
(all from Sigma Chemical Co.) and bisacodyl
metabolite, which was prepared as described.2
The evaluation was carried out independently in
two sites and the results compared. To evaluate
Correspondence: Dr A Duncan.
E-mail: Aduncan@gri-biochem.org.uk
TLC plates were prepared. The performance of
each system in detecting laxatives was scored as
7, +, ++, or +++ (poor, weak, moderate
or strong detectability). This subjective score
was based on the clarity of chromatographic
bands (vividness of colour, sharpness of band,
degree of colour change) and freedom from
interfering bands.
We initially used our routine laxative methods3
to evaluate ®ve different detection solutions:
6 mol/L sodium hydroxide,4 3 mol/L sodium
hydroxide, 3 mol/L potassium hydroxide,5 5%
potassium hydroxide in methanol:water (70:30)6
and Mandelin’s reagent.4 TLC plates were
sprayed for about 5±10 s from a distance of at
least 50 cm. Plates were also viewed under
ultraviolet (UV) light at 360 nm to monitor the
¯uorescence of anthraquinone laxatives.4
The performances of four commercially avail-
able TLC silica gel plates were compared: three
were silica gel high-performance thin-layer
chromatography (HPTLC) plates with con-
centrating zones (from: Whatman, Maidstone,
Kent; BDH, Poole, Dorset; and Merck, Poole,
Dorset, UK) and the last was a Schleicher and
Scheull TLC plate, used most frequently by
NEQAS participants (originally distributed by
Anderman and Co., Kingston-upon-Thames,
Surrey, UK, but not now available in the UK).2
Using the optimal TLC plate and detection
system, we compared 11 mobile-phase solvent
systems in current use by UK laboratories (see
Table 1). Six of these are described in the
literature.2±6
Lastly, the performance of solvent/solvent
extraction using chloroform/propan-2-ol (9/1)4
was compared with two solid-phase extraction
systems previously described4,5 (Extrelut NT20
prepacked columns from BDH and SepPak
C18 columns from Jones Chromatography,
Hengoed, Mid-Glamorgan, UK).

64
 TABLE 1. Comparison of mobile-phase solvent systems on the performance of laxative detection (listed in order of performance)

Detectability of laxatives

Mobile phase Rhein Aloe-emodin Emodin Phenolphthalein Phenolphthalein metabolite Bisacodyl metabolite

Ethyl acetate:toluene:glacial acetic acid (4:16:1)3 ++ + +++ ++ ++ ++

Hexane:toluene:glacial acetic acid (3:1:1)4 ++ + + ++ ++ ++
Chloroform:acetone (4:1)2 7 + + +++ +++ +
Dichloromethane:acetone (8:2) 7 + + ++ ++ ++
Ethyl acetate:methanol: water (100:17:3)6 ++ + + +++ 7 +
Ethyl acetate:methanol:concentrated ammonia (85:15:6) 7 + + ++ + +
Heptane:acetone:glacial acetic acid (50:50:0´2) ++ 7 ++ ++ 7 +
Hexane:4-methyl-2-pentanone*:glacial acetic acid + + + +++ 7 +
(20:20:1)3
Xylene:4-methyl-2-pentanone:methanol (10:10:1)4 + 7 7 ++ ++ 7
4-methyl-2-pentanone:xylene:hexane:glacial acetic acid + 7 + ++ 7 7
(60:20:12:0´5)5
Chloroform:acetone:glacial acetic acid:toluene 7 7 7 + 7 7
(40:10:20:10)

*4-methyl-2-pentanone is also known as methyl-isobutyl ketone and isopropyl acetone.

Evaluation of thin-layer chromatography methods for laxative detection

Ann Clin Biochem 2001: 38

65
66 Duncan & Phillips
RESULTS
Comparison of detection systems
The use of 5% methanolic potassium hydroxide
gave a poor colour response for all laxatives.
Spraying with aqueous solutions of sodium
hydroxide resulted in slightly clearer bands than
with potassium hydroxide. Little difference was
apparent between 3 mol/L and 6 mol/L sodium
hydroxide solutions, but with the latter bisacodyl
produced a slightly more intense colour response
and the phenolphthalein band faded rather more
slowly, and so this was the preferred option.
Anthraquinone bands produced an orange
¯uorescence at 360 nm, but the ¯uorescence was
not striking and was sometimes obscured by
other urinary pigments. We concluded that this
step did not confer any additional bene®t.
Similarly, spraying with Mandelin’s reagent did
not improve the detection of laxatives.
Bisacodyl metabolite is usually only detectable
following heating of the plate. We found that its
detection was made easier by visualizing the
development of the spot using a hair dryer or a
heating plate at >758C. The colour can be
intensi®ed further by placing the plate in an oven
at 1008C for 30 min.
Comparison of TLC plates
The performance of the three HPTLC plates in
laxative detection was signi®cantly better than
that of the Schleicher and Scheull plate, which
produced lighter and more diffuse spots. The
three HPTLC plates performed similarly; how-
ever, sample application was easier using What-
man HPTLC plates (LHP-KF, catalogue
number 4806410) in which the concentration
zone was less compacted.
Comparison of solid-phase extraction with solvent
extraction
No signi®cant differences in performance were
found for the three extraction systems. In terms
of practicability, Extrelut columns were the
simplest and quickest option. Although these
columns are initially expensive (approximately
£6´30 each), costs can be minimized by repack-
ing with loose Extrelut NT20 (from BDH) and
by scaling down urine volumes to 10 mL
(approximately £2´50 each).
Comparison of mobile-phase solvent systems
The choice of mobile phase had a marked effect
on the clarity of laxative bands (see Table 1).
There were signi®cant variations in the colour
produced, its intensity and stability, and
Ann Clin Biochem 2001: 38
potential interfering bands. Although there is
an element of subjectivity in reading TLC plates
there was good agreement when the results of
the two independent assessments were com-
pared. The best overall results were obtained
with ethyl acetate:toluene:glacial acetic acid
(4:16:1) and hexane:toluene:glacial acetic acid
(3:1:1), both of which correctly identi®ed the
laxatives in the positive urine samples. The
retention times (measured from the concentrat-
ing zone/chromatography zone intersect) found
for these two systems were: rhein 0´45 and 0´52,
respectively; aloe-emodin 0´34 and 0´32; emodin
0´55 and 0´4; phenolphthalein 0´32 and 0´00;
phenolphthalein metabolite 0´38 and 0´06; and
bisacodyl metabolite 0´15 and origin (i.e. within
the concentrating zone).
CONCLUSIONS
Perhaps the single most important factor in the
successful detection of laxatives by TLC is the
skill and experience of the operator. However,
methodological aspects contribute signi®cantly.
In this respect the choice of mobile phase is
particularly relevant, with ethyl acetate:tolu-
ene:glacial acetic acid (4:16:1) and hexane:tolu-
ene:glacial acetic acid (3:1:1) giving the best
results. The use of HPTLC plates with a
concentrating zone is recommended. There was
little to choose between solvent and solid-phase
extraction, although the use of Extrelut columns
may have practical bene®ts. Finally, visualiza-
tion under UV light or spraying with Mandelin s
reagent conferred little additional bene®t over
spraying with 6 mol/L sodium hydroxide and
subsequent heating.
REFERENCES
1 Duncan A. Screening for surreptitious laxative
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4 de Wolff FA, de Haas EJM, Verweij M. A screening
method for establishing laxative abuse. Clin Chem
1981; 27: 914±8
5 Perkins S, Livesey JF. A rapid high-performance
thin-layer chromatographic urine screen for laxative
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6 Maurer HH, Kraemer T. Rapid detection of anthra-
quinone laxatives in urine using high-performance
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Accepted for publication 3 October 2000