Introduction Paracetamol (4-acetoaminophen) has mild analgesic and antipyretic properties and is, along with acetylsalicylic acid, one of the most popular analgesic agents. The administration of this drug with caffeine yields analgesic effects significant- ly greater than that of paracetamol alone [1, 2]. Several methods have been employed for the spectrophotometric determination of this drug in combined dosage forms [3-7]. Chromatogra- phic methods have been proposed for the simul- taneous determination of paracetamol and other active principles [8-10], Fluorescence spec- troscopy methods were also described [11, 12], however those methods are time-consuming and use expensive equipments. Electroanalytical methods were proposed to paracetamol determi- nation in pharmaceutical formulations using car- bon paste electrode [13-15]. Flow injection systems are useful tools for the automation, miniaturization and simplifica- tion of analytical processes. These procedures permit obtain results faster and accurate than batch procedures [16]. Flow injection spectrophotometric sys- tems with spectrophotometric detection were described using nitrite as reactant [17-19]. Other spectrophotometric flow injection methods based on reaction of paracetamol to produce a indophenol dye [20-24] and systems with solid phase UV spectrophotometric detection [25-28] have been described for paracetamol determina- tion in pharmaceutical formulations. Flow proce- dures with chemiluminescence detection were described using luminol based reaction [29, 30] 47Ecl. Quím., São Paulo, 33(2): 47-54, 2008 www.scielo.br/eq www.ecletica.iq.unesp.br Volume 33, número 2, 2008 Spectrophotometric flow injection procedure to indirect determination of paracetamol in pharmaceutical formulations using o-tolidine as reagent O. Fatibello-Filho*, H. J. Vieira Departamento de Química, Centro de Ciências Exatas e de Tecnologia Universidade Federal de São Carlos, São Carlos, Brasil. *bello@dq.ufscar.br Abstract: A spectrophotometric flow injection method for the determination of paracetamol in pharma- ceutical formulations is proposed. The procedure was based on the oxidation of paracetamol by sodium hypochloride and the determination of the excess of this oxidant using o-tolidine dichloride as chro- mogenic reagent at 430 nm. The analytical curve was linear in the paracetamol concentration range from 8.50 x 10-6 to 2.51 x 10-4 mol L-1 with a detection limit of 5.0 x 10-6 mol L-1. The relative standard devia- tion was smaller than 1.2% for 1.20 x 10-4 mol L-1 paracetamol solution (n = 10). The results obtained for paracetamol in pharmaceutical formulations using the proposed flow injection method and those obtained using a USP Pharmacopoeia method are in agreement at the 95% confidence level. Keywords: paracetamol; pharmaceutical analysis; flow injection analysis; spectrophotometry. EQ07-AF 26/6/08 11:53 Page 47 and another method employed the oxidation of tris(2,2’-bipyridyl)ruthenium(II) by potassium permanganate [31]. A Fourier transformed infra- red (FTIR) method based on the detection of products formed in the alkaline hydrolysis of paracetamol was also proposed in the literature [32]. A flow-injection method with spectrofluori- metric detection to determine paracetamol based on its oxidation with hexacyanoferrate(III) was reported [33]. Different techniques for quantifica- tion of paracetamol in pharmaceuticals were evaluated in a recent review [34]. The major substances found as products of chlorination of paracetamol were 1,4-benzo- quinone and N-acetyl-p-benzoquinoneimine (NAPQI). Other products that were identified included the chloro-4-acetamidophenol and dichloro-4-acetamidopheno [35]. In this article a fast, simple and accurate flow injection procedure with spectrophotometric detection to determine paracetamol in pharmaceu- tical products exploiting the reaction between paracetamol and hypochlorite is proposed. In this method the excess of oxidant was determined spec- trophotometrically using o-tolidine (3,3’-dimethyl- benzidine) as reagent at a wavelength of 430 nm. Experimental Reagents and solutions All experiments were performed with chemicals of analytical grade: paracetamol (Sigma), o-tolidine hydrochloride (Merck), sodi- um hypochlorite (Vetec, São Paulo, Brazil) and sodium borate (Synth), were used as received. All solutions were prepared with desionized water from a Millipore (Bedford, MA) Milli-Q system (model UV Plus Ultra-Low Organics Water). A 8.53x10-3 mol L-1 paracetamol stock solution was prepared dissolving 129 mg of this drug with desionized water in a 100.0 mL cali- brated flask and reference solutions were pre- pared by appropriate stock solution dilution with desionized water. A 4.31x10-3 mol L-1 o-tolidine dichloride (3,3’-Dimethyl-(1,1’-biphenyl)-4,4’-diamine) stock solution was prepared dissolving 123.0 mg of the reagent with desionized water in a 100.0 mL calibrated flask. An additional dilution was performed in 250.0 mL calibrated flask with 0.19 mol L-1 HCl solution to obtain a 9.8x10-5 mol L- 1 o-tolidine work solution. A sodium hypochlorite stock solution was prepared diluting 2.0 mL of the concentrated solu- tion in a 250.0 mL calibrated flask with a 1.00x10- 2 mol L-1 borate buffer solution (pH 9.0). An addi- tional dilution was performed transferring 2.0 mL of this solution to a 250.0 mL calibrated flask and the volume was made up with a 1.00x10-2 mol L-1 borate buffer solution. This stock solution was standardized using a titrimetric method. A 0.19 mol L-1 HCl solution was prepared by dilution of 8.0 mL HCl (Merck) in 500.0 mL and this solution was standardized with a 0.100 mol L-1 NaOH standard solution. Brazilian pharmaceutical products such as, Cibalena® (Novartis Indústria Farmacêutica, S.B. dos Campos, SP); Tylenol 500®, Tylenol 750® and Tylenol DC® (Janssen-Cilag Farmacêutica, São José dos Campos, SP); Tyramol 750® (Labora- tórios Farmacêuticos Caresse, São Paulo); Parace- tamol 500® (EMS Sigma Pharma, Hortolândia, São Paulo); Resfry® (Laboratório Neo Química Com. Ind. Ltda, Anápolis, GO); Paracetamol® (Eurofarma Laboratórios Ltda, São Paulo) were purchased from a local drugstore and analyzed using the proposed flow method and a comparative USP Pharmacopoeial procedure [36]. Apparatus Flow injection spectrophotometric meas- urements were carried out using a Femto 485 spec- trophotometer (São Paulo, Brazil) equipped with a Hellma® flow cell (path length 10 mm, inner vol- ume 18 µL, NY, USA) connected to a Cole-Parmer (Chicago, Il, USA) model 1202-0000 two-channel strip-chart recorder. An Ismatec® IPC-12 peristaltic pump (Zurich, Switzerland) supplied with Tygon® tubing and an injector-commutator were used throughout [37]. The flow manifold was construct- ed with polyethylene tubing (0.8 mm i.d.). Preparation of pharmaceutical samples The contents of tablets or sachets were weighed and powered in a mortar. Mass varying from 120.0 to 156.0 mg of paracetamol were accu- rately weighed and dissolved with 30 mL of 48 Ecl. Quím., São Paulo, 33(2): 47-54, 2008 EQ07-AF 26/6/08 11:53 Page 48 desionized water in 50.0 mL calibrated flask. These solutions were gently shaken by 10 min and the volume was completed with desionized water. Another dilution from theses solutions were per- formed using aliquots varying from 195-200 µL to obtain final concentrations of ca. 1.10 x 10-4 mol L- 1 in 25.0 mL calibrated flask with desionized water. For the liquid formulation, an accurate aliquot of 200 µL was transferred to a 50.0 mL calibrated flask and the volume was completed with desionized water. The final concentration obtained was ca 1.1 x 10-4 mol L-1. Flow injection procedure In the flow injection manifold, shown in the Figure 1, when the all solutions were propelled by peristaltic pump, a baseline was generated by reac- tion between the sodium hypochlorite and the o- tolidine streams and continuously monitored by spectrophotometer at 430 nm (equation 1, Scheme 1). The sample or standard solutions containing paracetamol (80 cm; 400 µL) were injected with the aid of injector-commutator into desionized car- rier stream (C; 2.2 mL min-1) and merges down- stream with 9.4x10-5 mol L-1 hypochlorite solution in 1.00x10-2 mol L-1 borate buffer solution (pH 9.0) (R1; 0.9 mL min-1) in the confluence point X pro- moting the consumption of ClO- (equation 2, Scheme 1). After the reaction coil RC1 (75 cm), the dispersed sample zone merges with a colorless 9.8x10-5 mol L-1 o-tolidine solution in 0.20 mol L- 1 HCl solution at confluence point Y (R2; 1.6 mL min-1) where the yellow reaction product was mon- itored spectrophotometrically. In this way, the decrease of absorbance (∆A) caused by hypochlo- 49Ecl. Quím., São Paulo, 33(2): 47-54, 2008 Figure 1. Flow injection spectrophotometric method for paracetamol determination in pharma- ceutical products. PP: peristaltic pump; L: sample loop (400 µL); C: carrier (H2O; 2.2 mL min-1); R1: 9.4x10-5 mol L-1 hypochlorite solution (0.9 mL min- 1); R2: 9.8x10-5 mol L-1 o-tolidine solution (1.6 mL min-1); I: injector-commutator (80 cm; 400 µL); RC1 and RC2: reaction coil (75 cm, both); D: spec- trophotometer (λ = 430 nm); X and Y: confluence point and W: waste. The distance between the injector I and X confluence point was 5 cm. rite consumption was proportional to concentration of paracetamol in the solution injected. Comparative procedure To validation of proposed flow injection method the results obtained were compared with those results obtained using a comparative USP Pharmacopoeia procedure [36]. This procedure is based in the intrinsic absorption of paracetamol in UV region (λ = 244 nm). For the determination of paracetamol, the samples and reference solu- tions were diluted in sodium hydroxide solution and the concentration of paracetamol was per- formed by interpolation in an analytical curve. Scheme 1. EQ07-AF 26/6/08 11:54 Page 49 Results and discussions Paracetamol exhibited significant reactivity with hypochlorite when exposed in large molar excess, leading to the production of multiple prod- ucts such as 1,4-benzoquinone, N-acetyl-p-benzo- quinoneimine and another products with molar mass higher than 300 g mol-1 [12, 35]. The proposed flow injection system is based on the ability of oxidation of paracetamol by hypochlorite in excess at pH 9.0 and the hypochlo- rite excess was monitored with o-tolidine produc- ing a yellow diiminequinone at pH 9.0 [12, 38]. The chemical and flow injection parame- ters were optimized by univariated method in order to achieve a good compromise between the peak height, reproducibility, accuracy, precision, baseline stability and analytical frequency. Chemical parameters Preliminary studies to optimize chemical parameters were carried out using a manifold shown in Figure 1. In this flow system the hypochlorite and o-tolidine flow rates was 1.2 mL min-1 and that of carrier was 2.2 mL min-1, the sample loop of 100 µL, reaction coil length RC1 of 150 cm and reaction coil length RC2 of 60 cm were also used. Initially, the effect of hypochlorite concentration over the range from 2.4x10-5 to 1.2x10-4 mol L-1 on the analytical signal (∆A) for a 9.4 x 10-5 mol L-1 paracetamol solution, using a 9.8 x 10-5 mol L-1 o-tolidine in 0.22 mol L-1 HCl solution was investigated. The analytical signal increased with increase of hypochlorite concentration up to 9.4x10-5 mol L-1 and, remaining constant for higher concentrations. Thus, a 9.4 x 10-5 mol L-1 hypochlorite solution was selected in further work. The effect of HCl concentration in the o- tolidine solution on the peak high was studied from 4.4 x 10-2 to 0.22 mol L-1 using a 9.4 x 10-5 mol L-1 hypochlorite solution and 9.8 x 10-5 mol L- 1 o-tolidine solution. The analytical signal increased with increase of the HCl concentration up to 0.20 mol L-1 and remaining constant for higher concentrations. Thus, the 0.20 mol L-1 HCl was selected for further work. To study the effect of pH of hypochlorite solution, a 1.00x10-2 mol L-1 borate buffer solution was employed. The study was performed over the pH range from 7.0 to 11.4. The analytical signal showed a slight increased with increase of pH up to pH 9.0. Higher pH promotes a severe interfere in the reaction between ClO- and o-tolidine decreas- ing the analytical signal due the consumption of HCl of the stream before the confluence point. The effect of o-tolidine concentration on the analytical signal was investigated in the con- centration range from 1.6 x 10-5 to 1.2 x 10-4 mol L-1. It was observed that the analytical signal increased with the increases of o-tolidine concen- tration up to 9.8 x 10-5 mol L-1 remaining constant for higher o-tolidine concentrations. Furthermore, a 9.8 x 10-5 o-tolidine solution was selected for fur- ther studies. Flow injection parameters The effect of the coiled reactor (RC1 and RC2) length on the analytical signal was studied from 30 to 150 cm. In this study, it was observed that increasing the length of coiled reactor RC1 up to 75 cm promote a slight increase of analytical signal. For higher coiled reactor length the analyt- ical signal decreased. Therefore, a 75 cm reactor length (RC1) was selected. The effect of reactor coil RC2 length was studied over the same range, the signal showed best peak height using a 75 cm length. A reactor coil RC2 of 75 cm length was selected to further experiments. The effect of sample volume from 100 to 600 µL on peak height (∆A) was studied for a 5.8 x 10-4 mol L-1 paracetamol reference solution. The analytical signal increased with increase of sample loop volume. Considering the repeatability and height of analytical signal, a 400 µL sample loop length was selected. The effect of flow rate for o-tolidine, hypochlorite and carrier (desionized water) on ana- lytical signal was studied in the flow rates of 0.9, 1.6 and 2.2 mL min-1, respectively. It was found that the analytical signal decreased deeply with the increasing of hypochlorite flow rate (R1). Therefore, a flow rate of 0.9 mL min-1 was select- ed for further experiments. In the o-tolidine flow rate (R2) study it was observed that the highest ana- lytical signals were obtained for the 1.6 mL min-1 flow rate. This flow rate was selected as optimum. In the study of effect of carrier flow rate on the ana- lytical signal showed that the flow rate of 2.2 mL 50 Ecl. Quím., São Paulo, 33(2): 47-54, 2008 EQ07-AF 26/6/08 11:54 Page 50 min-1 promotes the best analytical signal in terms of sensitivity and analytical frequency. Thus, a flow rate of 2.2 mL min-1 was selected in this work. Interferences and recovery studies The selectivity of the proposed flow injec- tion procedure was studied comparing the analyti- cal signal of a 1.5 x 10-4 mol L-1 paracetamol stan- dard solution with 1.5 x 10-4 mol L-1 paracetamol reference solutions containing the commonly excipients found in commercial pharmaceutical formulations in the concentrations that cause a error of ±5%. The studied excipients were 51Ecl. Quím., São Paulo, 33(2): 47-54, 2008 Table 1. Recoveries of paracetamol standard solu- tions. Samples Added / Founded / Recovery / 10-4 mol L-1 10-4 mol L-1 % Cibalena® 0.50 0.50 100 0.95 0.96 101 1.12 1.13 101 Tylenol 500® 0.50 0.50 100 0.95 0.93 97.8 1.12 1.09 97.3 Tyramol 750® 0.50 0.51 102 0.95 0.97 102 1.12 1.10 98.2 Tylenol 750® 0.50 0.52 104 0.95 0.97 102 1.12 1.11 99.1 Paracetamol 500® 0.50 0.50 100 0.95 0.97 102 1.12 1.15 103 Tylenol DC® 0.50 0.50 100 0.95 0.94 98.9 1.12 1.10 98.2 Resfry® 0.50 0.50 100 0.95 0.94 98.9 1.12 1.10 98.2 Paracetamol® 0.50 0.49 98.0 0.95 0.94 98.9 1.12 1.11 99.1 n=3. polivinylpirrolidine, citric acid, saccharine, tar- trazine, caffeine, EDTA and sodium car- boxymethylcellulose. Only the citric acid and caf- feine showed high interference above an analyte:interference ratio of 1:10. Probably, these substances are oxidized by ClO-. But, the amounts of theses substances in the commercial pharma- ceuticals are lower than that of paracetamol. The other excipients do not shown any interference at the same concentration of paracetamol. The study of addition and recovery of paracetamol in pharmaceutical products was per- formed to verify the interference in potential of sample matrix. Tablets and syrups were employed. This study was performed spiking three aliquots of paracetamol standard solution in the concentra- tions of 5.00 x 10-5; 9.50 x 10-5 and 1.10 x 10-4 mol L-1 in a sample solution containing 1.12 x 10-4 mol L-1 paracetamol. Recoveries of paracetamol ranged from 97.3 to 103% as shown in Table 1. These results obtained suggested an absence of matrix effect on those determinations. Analytical features and application The flow injection procedure developed present an analytical curve linear in the paraceta- mol concentration range form 8.50 x 10-6 to 2.51 x 10-4 mol L-1 described by equation: ∆A = 0.058 + 1.571,41 x C; r = 0.9990, where ∆A is (A1 – A2), the difference between the baseline absorbance (A1) and the transient signal of solution injected containing paracetamol, and C is the paracetamol concentration in mol L-1. The relative standard deviation (RSD) was lower than 1.2% (n = 10) for 1.20 x 10-4 mol L-1 paracetamol solution and the detection limit obtained was 5.0 x 10-6 mol L-1 (three times blank signal/inclination of analytical curve). The analyt- ical frequency of 60 determinations per hour was obtained. The paracetamol determination was per- formed using the developed spectrophotometric flow injection method and the results obtained were compared with those obtained using a USP Pharmacopoeia procedure [36]. As shown in Table 2, the results obtained using the proposed flow injection procedure are in good agreement with those results obtained using the comparative pro- cedure at confidence level of 95%. EQ07-AF 26/6/08 11:54 Page 51 Analytical features attained by the pro- posed flow procedure and those reported by described flow procedures with spectrophoto- metric detection are presented in Table 3. The lin- ear response was comparable with flow system were paracetamol was hydrolyzed before the detection [39] and higher than system described were paracetamol is monitored at an optode [18, 27]. The procedure proposed by Knochen et al. [17] uses a second degree polynomial analytical curve. The sampling rate was higher than proce- dures that have used alkaline hydrolysis and optode as sensor. The detection limit was better than previously reported by Burakham et al. [18], 52 Ecl. Quím., São Paulo, 33(2): 47-54, 2008 Table 2. Determination of paracetamol in pharma- ceutical products using the flow injection and the USP procedure [36]. Samples Paracetamol/mg g-1 Relative error USP Proposed (Er / %) Cibalena® 300 ± 5 302 ± 3 0.7 Tylenol 500® 850 ± 4 840 ± 4 -1.2 Tyramol 750® 860 ± 6 820 ± 3 -4.6 Tylenol 750® 850 ± 7 861 ± 4 1.3 Paracetamol 500® 820 ± 4 868 ± 3 5.8 Tylenol DC® 760 ± 6 771 ± 2 1.4 Resfry® 410 ± 5 391 ± 1 -4,6 Paracetamol®(*) 198 ± 6 203 ± 2 2.5 n=3; (*)mg L-1; Er: FIA vs. USP procedure. Table 3. Analytical features of spectrophotometric flow procedures for paraceta- mol determination in pharmaceuticals. a not reported. but compare unfavorably with those obtained by Criado et al. [39] and by Cañada et al. [27]. However, these flow procedures have showed lower sampling rate. The proposed flow system compare favor- ably with a chemiluminescence flow procedures described by Alapont et al. [29] and by Pulgarin and Bermejo [33], showing better precision and largest linear range. Conclusions The proposed flow procedure was suitable for the determination of paracetamol in pharma- ceutical formulations, presenting accuracy and precision necessary to quality control laboratories. The paracetamol determination not requires exten- sive preliminary sample treatment. The procedure does not require expensive reagents and can be implemented easily in pharmaceutical laborato- ries. This procedure is simpler than spectrofluori- metric procedure described by Vilchez et al. [12] due the use of a suitable spectrophotometer. Acknowledgements The scholarship granted to H.J.V. from CAPES is gratefully acknowledged. Received 08 February 2008 Accepted 28 May 2008 EQ07-AF 26/6/08 11:54 Page 52 References [1] L.S. Goodman, A. Gilman, Bases Farmacológicas de la Terapêutica, 4 edition, New York, Interamericana, 1974. [2] F.D. Fuchs, L. Wannmacher, Farmacologia Clínica - Fundamentos da Terapêutica Racional, 2 ed., Rio de Janeiro, Guanabara Koogan, 1999. [3] E. Dinc, C. Yucesoy, F. 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O desvio padrão relativo foi menor que 1,2% para uma solução de paracetamol de 1,20 x 10-4 mol L-1 (n=10). Os resulta- dos obtidos nas análises de produtos farmacêuticos comerciais obtidos empregando o procedimento desen- volvido foram comparados com aqueles obtidos empregando o procedimento descrito na Farmacopéia Americana (USP XXII) estando em concordância ao nível de confiança de 95%. Palavras-chave: Paracetamol; produtos farmacêuticos; análise por injeção em fluxo; espectrofotometria. EQ07-AF 26/6/08 11:54 Page 53