SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

C30
SIMULTANEOUS DETECTION OF STIMULANT LAXATIVES AND DIURETICS IN HUMAN
URINE USING GC-MS AFTER ENZYMATIC CLEAVAGE OF CONJUGATES AND
EXTRACTIVE METHYLATION
J. Beyer', A. BierI, F.T. Peters and H.H. Maurer
Department of Experimental and Clinical Toxicology, University of Saarland
0-66421 Homburg (Saar), Germany,jochen.beyer@uniklinik-saarland.de
Background: Laxatives and diuretics are widely abused for various reasons. Surreptitious abuse of both is
often associated with eating disorders or Milnchhausen syndrome. Concerning laxatives, there is also
habitual abuse because of chronic constipation, whereas diuretics are also abused in doping. Such abuse
may lead to serious disorders like chronic diarrhea, hypokalemia, dehydration, and disturbance of acid-base
balance. Because of the heterogeneity of these side effects and their similarity to symptoms of
gastrointestinal or renal disorders, a toxicological screening for laxatives and diuretics should be part of the
differential diagnosis of such syndromes. This may also help to avoid extensive and expensive diagnostic
work.
Methods: After accelerated enzymatic cleavage of conjugates (glucuronidase/arylsulfatase, EC no.
3.2.1.3113.1.6.1, 100000 Fishman units per mL, 50 cC, 90 min), the drugs and their metabolites were
isolated from 2 mL of urine by extractive methylation. For details of extractive methylation see Maurer HH
et aI., JAT 25,2001,237. The extract was reconstituted in 50 I-lL ethyl acetate and 2 J..lL were injected into
the GC-MS (Agilent GC-MSD 5972). Analytes were separated on an HP 1 column (12 m x 0.2 mm 1.0.)
and detected by mass-spectrometry in the EI full scan mode. They were screened by using reconstructed
mass chromatography using selective ions and identified by visual and computerized comparison of the
peak underlying mass spectra with the corresponding reference mass spectra (PMW _tox4).
Results: The assay allowed the detection of the diphenylmethane laxatives phenOlphthalein, bisacodyl and
picosulfate (the latter two via their common metabolites bisacodyI diphenol, methoxy bisacodyl diphenol
and dimethoxy bisacodyldiphenol) as well as of anthraquinone laxatives contained in plants like senna,
cascara, rhubarb, frangula and aloe via their common metabolite rhein. Furthermore, the diuretics
acetazolamide, bemetizide, bendroflumethiazide, bumetanide, butizide, canrenoic acid (also main
metabolite of spironolactone), carzenide, chlorothiazide, chlortalidone, c1opamide, cyclopenthiazide,
cyclothiazide, diclofenamide, etacrynic acid, etozolin, furosemide, hydrochlorothiazide, indapamide,
mefruside, metolazone, piretanide, poly thiazide, tienilic acid and xipamide could be detected as well as the
uricosurics benzbomarone, probenecide and sulfinpyrazone, which are relevant in doping control. Cleavage
of conjugates was a prerequisite for sensitive detection of the laxatives and/or their metabolites, because
these analytes are excreted mainly as conjugates. For analysis of diuretics, enzymatic hydrolysis is not
necessary, because they are mainly excreted unchanged. The limits of detection (LOD, SIN 3) ofthe tested
drugs and their metabolites lay in the range of I to 500 nglmL, 90% within 5 to 100 nglmL. Recoveries
were determined for a limited number of analytes (bisacodyl diphenol, butizide, furosemide,
hydrochlorothiazide, phenolphthalein, piretanide, probenecide, rhein, sulfinpyrazone, and tienilic acid)
representing the different groups of the structurally heterogeneous analytes and ranged from 33 to 99 with
coefficients of variation from 4.2 to 21.1 % (determined at 5 x LOD). Applicability studies showed that at
least the given drugs and/or their metabolites were detectable over periods of 24 to 72 hours after
administration of the lowest therapeutic dose (picosulfate, 5 mg; furosemide, 40 mg; hydrochlorothiazide,
15 mg; spironolactone, 25 mg; or senna plant extract containing 7 mg ofsen nos ide B) to one young healthy
volunteer each (after informed consent). Furthermore, the extractive methylation (without the enzymatic
cleavage) has proved to be suitable for simultaneous detection of other acidic drugs (Maurer HH, J.
Chromatogr. B 733, 1999, 3).
Conclusion: The presented GC-MS procedure allowed simultaneous identification and differentiation of
stimulant laxatives, diuretics and/or their metabolites in urine. This procedure should be applicable for
diagnosis or differential diagnosis of an abuse ofthe described drugs or for doping control.
Keywords: Diuretics, Laxatives, GC-MS
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