A simple urine test for sulfonamides

Bulletin ofthe WorldHealth Organization, 61(1): 167-168(1983) ©3 World Health Organization 1983 A simple urine test for sulfonamides JUVENAL DE ALMEIDA-FILHO' & JOSE MARIA DE SOUZA2 The lack of a simple anrd reliable testfor sulfonamides has created a problem because of the increasing use of these drugs in the chemotherapy ofmalaria. This paper describes a modification of the Bratton-Marshall technique, for which the reagents are easily obtain- able and which can be carried out in simply equipped primary health care laboratories. A prerequisite for the conduct of clinical trials of antimalarial drugs and the interpretation of in vitro and in vivo tests for determining the drug sensitivity of Plasmodium falciparum is a simple and reliable method of assessing whether a patient has received medication prior to the trial or the performance of the test. As such tests have to be conducted under field conditions, a simple urine test is preferable. Such a test does exist for 4-aminoquinolines (4) but the increasing use of sulfonamides, usually in combi- nation with pyrimethamine, and the poor results often obtained by the lignin test for sulfonamides (3) necessitated the development of a more reliable test for these drugs. Feigl & Anger in 1975 described a modification of the reliable Bratton-Marshall reaction for sulfon- amides (1) and produced a very sensitive and simple spot test on filter paper, but the reagent used is neither readily available nor listed in the current chemical catalogues (2). This paper describes another simple modification of the Bratton-Marshall technique, for which the reagents are obtainable and which can be carried out in simply equipped peripheral labora- tories. MATERIALS AND METHODS Reagents Reagent 1: sodium nitrite solution, 0.03 mmol/l or 0.2 mg/100 ml or 0.2 Vo (w/v) in dis- tilled water. Reagent 2: hydrochloric acid (concentrated). Reagent 3: Bratton-Marshall solution (BM); dis- solve 20 mg N-(l-naphthyl)-ethylene diamine dihydrochloride in 20 ml dis- tilled water, adding 3 drops of concen- trated hydrochloric acid. Chief, Analytical Laboratory, Q.C. Department, Hoffmann- La Roche Laboratories, Rio de Janeiro, Brazil. 2 Hospital Barros Barreto, Belem, Para State, Brazil. Reagents 1 and 2 are stable at room temperature. Reagent 3 is stable for at least 2 months if kept at 5-10 °C in a brown bottle. Procedure (i) Pipette 1 ml of urine into a test tube. (ii) Add 1 drop of the sodium nitrite solution (reagent 1) and 2 drops of concentrated hydrochloric acid and mix well. Let it stand for 1 minute. (iii) Add 3 drops of BM (reagent 3) and mix. RESULTS The evaluation of the test is equally simple: - it is positive for free sulfadoxine and other diazotizable arylamines when the solution possesses a persistent purple colour. If the test is to be read spectrophotometrically, optimum absorbance is at 540 nm; - the test is negative if the solution does not become purple or if the purple colour quickly dis- appears and the solution becomes greenish or brownish. DISCUSSION The test has been evaluated in the laboratory and in the clinic where the urine of 150 patients taking Fansidar ® (sulfadoxine and pyrimethamine) was tested for the presence of sulfonamide. It has been shown to give reliable results and to be sensitive down to a sulfonamide concentration of 16 Amol/l of urine (or 5 mg/l or 0.5 mg/%o); the lower the sulfonamide content of the urine, the longer it takes for the colour reaction to develop (30 seconds to 30 minutes). False positives are obtained with nitrazepam, clon- azepam, flunitrazepam and other drugs which carry an aromatic primary amino group or a drug which is metabolized to such compounds. 4273 -167- 168 J. DE ALMEIDA-FILHO & J. M. DE SOUZA In the original Bratton-Marshall test, sulfamate was used to remove the excess nitrous acid between steps (ii) and (iii) of the procedure, but Wehrli (5) has suggested that this could be overlooked. We found that such removal was, indeed, essential; however, extensive laboratory and field trials indicated that the excess nitrous acid was removed by urea which is always present in the urine at concentrations that were always many times greater than the concentrations of sulfamate originally used. ACKNOWLEDGEMENTS We are grateful for the assistance of Dr Ivo Ferreira of Hoffmann-La Roche, Rio de Janeiro. This study received partial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. RESUME UN TEST SIMPLE DE DtTECTION DES SULFAMIDES DANS L'URINE Pour executer les essais cliniques des medicaments anti- paludiques et interpreter les epreuves in vitro et in vivo de determination de la pharmacosensibilite de Plasmodium falciparum, une condition prealable est de disposer d'une methode simple et fiable permettant d'estimer si un malade a requ un traitement avant l'essai ou l'execution du test. L'absence d'un tel test pour les sulfamides etait devenue un probleme en raison de l'utilisation croissante de ces medica- ments. Une epreuve sur l'urine, fondee sur la technique de Bratton-Marshall, a e mise au point pour les sulfamides. Ce test necessite trois reactifs: 1) une solution de nitrite de sodium (0,03 mmol/l ou 0,2 mg/100 ml dans de l'eau distillee), 2) de l'acide chlorhydrique (concentre), 3) de la solution de Bratton-Marshall (obtenue en dissolvant 2 mg de dichlorhydrate de N-(napthyl-l)-ethylenediamine dans 20 ml d'eau distillee, et en ajoutant 3 gouttes d'acide chlorhydrique concentre). Ces reactifs sont faciles a se procurer a la difference de ceux qui etaient utilises dans le test de Bratton-Marshall original. Le test lui-meme est simple a executer et a evaluer et il peut etre effectue dans des laboratoires peripheriques ne disposant que d'un materiel simple. Il a et montre qu'il donne des resultats fiables et qu'il est sensible A une concentration de sulfamides aussi faible que 16 jmol/l d'urine (ou 5 mg/l). Des resultats faussement positifs sont obtenus avec le nitrazepam, le clonazepam, le flunitrazepam et d'autres medicaments qui comportent un groupement amine primaire aromatique ou des medicaments qui sont metabolises en de tels composes. REFERENCES 1. BRATTON, A. C. & MARSHALL, E. K. Journal of biological chemistry, 128: 537 (1939). 2. FEIGL, F. & ANGER, V. Spot tests in organic analysis, 7th ed., Amsterdam, Elsevier, 1975, pp. 643-644. 3. HEPLER, 0. E. Manual of clinical laboratory methods, 4th ed. Springfield, Illinois, C. C. Thomas, 1973, p. 21. 4. LELIJVELD, J. & KORTMANN, H. Bulletin of the World Health Organization, 42: 477-479 (1970). 5. WEHRLI, A. In: Stahl, E., ed., Thin layer chroma- tography, 2nd English edition. Heidelberg, 1969, p. 543.