Urinary Excretion of Paracetamol A Thin-Layer Chromatography Exercise for Nursing Students Raymond J. S. Hickman School of Nursing, Flinders University of South Australia, GPO Box 21 00, Adelaide 5001, Australla Thin-layer chromatography (TLC) is a remarkable technique. It combines economy, versatility, and technical and conceptual simplicity. I t can be used to good effect in settings as different as a research laboratory and a junior teachine .. laboraton. This DaDer . . describes its use by nursing students in a faboratory exercise designed to &firm some im~ortantfeatures of the usual textbook description of drug metabolism and excretion. Most drugs are excreted in urine either unchanged or as metabolites. These metabolites are usually formed in the of the drug liver and oRen by" coniupation " - molecule with an endogenous substance such as g l u c u r o ~ cacid. Conjugation has a ~rotectiveeffect, because it produces substances that are less toxic, and &ore soluble in water, than the original drug. The lower toxicity reduces the adverse effects that the drug has while it remains in the body and the higher solubility in water increases the rate a t which it is cleared from the body as a component of urine. The conjugated form of a drug often can be hydrolyzed to re-form the original drug. Hydrolysis may be achieved by simple chemical means, such as heating with a strong acid, or by use of a suitable enzyme. Where an enzyme is used for hydrolysis the identity of the enzyme that regenerates the original drug will reveal the type of metabolite present. In this exercise the enzyme P-glucuronidase is used to investigate the metabolism of paracetarnol (also known as acetaminophen or p-hydroxyaceta~lide). Methods Thin-layer chromatography was carried out on commercially prepared (Merck)plastic sheets coated with silica gel HF25a. Each chromatography plate was a 80 x 50 mm section cut from one of these sheets. Samples were applied to the plate using calibrated micropipets. The developing solvent was a mixture of butyl acetate, chloroform, and 90% formic acid (9:4:1). Because 90% formic acid is capable of causing contact bums to tissue and produces fumes that are imtating to the skin, eyes and respiratory system ( I ) , the developing solvent should be prepared in a fume cupboard.
'CAUTION: Safety glasses and protective gloves should be worn during this operation. Plates were developed in glass jars fitted with tight-fitting, screw-down lids, and the developed plates were dried in a fume cupboard using a hair drier. In this way the concentration of organic solvent in the atmosphere of the laboratory was kept to a minimum throughout the practical sessions. Spots were detected by viewing the plates under ultraviolet light of wavelength 254 nm. The p-glucuronidase was from mollusks and was supdied bv BDH. The ethvl acetate used for liauid-liauid extraction was of analytical grade. The buffer solution used was NaH9P0,. 0.2 mu1 L-'Ma9HPO3.0.2 mol L-' 193:7. DH = 5.5). he piacetamol inges&d wasin the form of 500 k g tablets (Panamax by Winthrop). Urine samples were processed in 100 x 16mm sample tubes fitted with screw caps and manufactured by Wheaton.
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Journal of Chemical Education
Experiment Urine was collected on two successive mornings from a human volunteer. On the first occasion no paracetamol had been ingested the previous evening and the urine obtained was used in the preparation of "normal" samples. On the second occasion, the volunteer had ingested 1g of paracetamol prior to retiring, and the urine obtained was used in the preparation of "test" samples. A set of normal urine samples was prepared by mixing 3-mL aliquots of the normal urine with 2-mL aliquots of buffer solution in separate sample tubes. Two sets of test samples were prepared. The first set was obtained by mixing 3 mL of test urine with 2 mL of buffer solution and then adding 1mg of p-glucuronidase to each sample tube. The second s e t was identical to t h i s except t h a t Pglucuronidase was not added. All three sets of sample tubes were then placed in a thermostat bath, a t 37 'C, and agitated gently overnight. They were then ready to be used by students in the laboratory session. Students worked in groups of three and subjected each urine sample to extraction with ethyl acetate (1x 2 mL) by vortex mixing for 1min. This led to the formation of emulsions that were broken by centrifugation. Thin-layer chromatographicexammation ofthe ethyl acetate laver in each test tube was carried out in the usual manner (2.a n d a m parison made with a solution of authentic paracetamol. Results and Discussion Although very few of the students attempting this exercise had had any previous experience with TLC, most of them were able to obtain a convincing result a t this first attempt to use the technique. Under the chromatographic conditions used, the &value observed for paracetamol was 0.38. In an account of student laboratory work published previously in this Journal (31, it was reported that paracetamol was not detectable by TLC in the urine of people who had ineested the comoound. In the Dresent case. however. the test sample containing no P-glu&ronidase displayed a small. faint. but unmistakable ~aracetamol. . spot . showing that some of the compound reaches the urine unchanged a s is reported in the original literature dealing with paracetamol metabolism. Thus, Prescott reports (4)that 5% of a nontoxic dose of paracetamol is excreted unchanged in the urine of humans. It is likely that the ethyl acetate extraction used in the present procedure is the reason this small level of paracetamol can be detected so readily by the TLC technique. The extraction produces a "cry riean sample in the sense that only a smnllam(~untof the normal urine components that ahsorb ultraviolet radiation of wavelength 254 nm is extracted along with the paracetamol. None of this material appears to shift from the original spot during development of the TLC plate and so the region of the plate in which paracetamol spots are detected,-after de\dnpment, is completely free of ultra\iolet-abwrbing substances other than parncetamol itself.
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The most striking feature of the TLC plate is the difference between the two test samples. The one to which Pglucuronidase has been added displays a large, intense paracetamol spot, but, as already stated, the one without P-glucuronidase shows only a small, faint spot. This difference serves as the main focus for discussion bv students. They are expected to consider the difference and then account for it in terms of the presence of a glucuronide metabolite in the urine and the action on that metabolite of
niques. When the technically sophisticated technique of Hi~h Pressure Liauid Chromatoera~hv(HPLC) is used to acgieve the same bbjectives as t k s ?Lk'exercise the com~lexitvof the eaui~mentbecomes an obstacle to understanding for many students. In contrast to this, the TLC exercise is well received by students, and it effectively illuminates what would otherwise be a piece of science simply read about in textbooks. Literature Cited
Conclusion
The technical and conceptual simplicity of thin-layer chromatography makes it an ideal technique for use by students having little experience with chemical tech-
1.Muir. G. T Ed.Hmerds in f h Chomiml Lobomlorv 2nd 4.:The Chemical Soeiehi:
Inndon, 1977. 2. Lindmmberg, W W;Bsumgarten, H . E . Ogonle Exprimon*l6th d.; D. C . Heath and Company k i w h n , MA. 1987. S.CorKll, J.A.J. Chem.Edu.1988,65,551-553. 4.Pnscott, h F BrU. J. C l i n Phormoeol. 1980.1WSupp. 21.291-2988
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