DRUGS, COSMETICS , FORENSIC SCIENCES Determination of Salicylate in Blood Serum by Flow Injection with Immobilized Salicylate Hydroxylase MARTA M.D.C. VILA and MATTHIEU TUBINO Universidade Estadual de Campina, Instituto de Química, C.P. 6154, 13083-970 Campinas, São Paulo, Brazil GRACILIANO DE OLIVEIRA NETO Universidade São Francisco, Centro de Ciências Biológicas e da Saúde, São Paulo, Brazil A flow injection (FI) enzymatic system, based on the use of immobilized salicylate hydroxylase in glass beads, was developed for the determination of salicylate. Salicylate hydroxylase and nicotinamide adenine dinucleotide (NADH) are used to convert salicylate to catechol. The reaction of catechol with 4-aminophenol at high pH yields a colored product which is detected spectrophoto- metrically at 565 nm. Ten samples of human serum containing from 5.0 ´ 10 –4 to 5.0 ´ 10 –3 mol/L added salicylate were analyzed and the recovery was de- termined. Eight additional serum samples contain- ing salicylate were analyzed by the Trinder test and the proposed method. The results obtained with the 2 methods showed good agreement by the sta- tistical Student’s t-test. The relative precision of the method is about 3.4% (RSD of the mean recov- ery). Considering the lowest concentration ana- lyzed, the quantitative limit of detection is about 0.2 ´ 10 –5 mol/L (3 ´ SD). The volume of the sample used was 150 mL. The proposed method was also used to analyze medicines containing acetylsalicylic acid. The results were statistically compared with those obtained through the U.S. Pharmacopoeia procedure and showed excellent agreement. A cetylsalicylic acid (aspirin), introduced in medicine in 1899, is still one of the most commonly used analge- sic, antipyretic, and anti-inflammatory drugs. In a new therapeutic use, based on its antiplatelet aggregation property, aspirin is used to treat cardiovascular complications (1). Most of the absorbed aspirin reaches the systemic circulation as salicylate anion, reaching its maximal level in the blood serum 2 h after ingestion (2). Therefore, plasma levels of salicylate are measured in patients to assess the effectiveness of therapy and to avoid side or toxic effects during long-term treatment. Short-term analgesic/antipyretic doses of aspirin (25–50 mg/kg per day) produce only relatively low serum salicylate concentrations (30–100 mg/L) and almost never re- quire therapeutic monitoring. In contrast, long-term anti-inflammatory doses of aspirin, which are given primarily to patients with various forms of arthritis, generally require laboratory monitoring to maintain serum salicylate within the therapeutic range, 150–300 mg/L serum. Because the differ- ence between therapeutic and toxic dosages is relatively small, a rapid and specific method is needed to determine salicylate levels in the blood. Severe symptoms can be ob- served when salicylate levels in serum are >300 mg/L (2, 3). Various methods for determining salicylate, which have been reported in the literature, make use of a large variety of analytical techniques. High-pressure liquid chromatography and gas–liquid chromatography (4–10), fluorescence (11, 12), and ultraviolet spectrophotometry (13) have been used. The methods are highly sensitive, but many of these techniques re- quire time-consuming sample pretreatment and are not suit- able for use in emergency situations where a rapid test is re- quired. Potentiometry analysis using ion-seletive electrodes (ISEs) has been used to determine salicylate in pharmaceutical samples (14, 15) and blood serum (16). Common problems with the ISEs are interferences from anions normally present in biological fluids. In the clinical laboratory, colorimetric techniques are most popular and, of these, the Trinder test (17) is the most commonly used. The method is based on the for- mation of a colored complex when salicylate and iron(III) ions are combined. However the purple color in the weakly acid solution is nonspecific. A color reaction is obtained with a wide range of compounds found in body fluids, particularly phenols and aliphatic enols. For a more specific analysis, an enzymatic method is used (18). The hydroxylation of the salicylate is catalyzed by salicylate hydroxylase (E.C. 1.14.13.1.), and this reaction can be used for analytical pur- poses. Although the enzymes are expensive and may increase the cost of the analysis, immobilization of the enzyme for re- peated use significantly reduces the cost. The immobilization can be done on solid supports which are then packed in reac- tors (immobilized enzyme reactor; 19) or in close proximity to an electrode surface (20–22). This study describes a flow injection (FI) system with colorimetric detection for determination of salicylate in serum samples and in medicines. It is based on the generation of catechol by the passage of samples with salicylate and nicotinamide adenine dinucleotide (NADH) through a reactor VILA ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1363 Received February 24, 2000. Accepted by JM November 23, 2000.