Indirect spectrophotometric determination of p-aminobenzoic acid in sunscreen formulations by sequential injection analysis

Analytica Chimica Acta 493 (2003) 233–239

Indirect spectrophotometric determination of p-aminobenzoic acid in sunscreen formulations by sequential injection analysis A. Salvador a,∗ , A. Chisvert a , A. Rodr´ıguez a , J.G. March b a

Departamento de Qu´ımica Analitica, Facultad de Qu´ımica, Universitat de València, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain b Departamento de Qu´ımica, Facultad de Ciencias, Universitat de les illes Balears, Carretera de Valldemossa, km 7.5, 07071 Palma de Mallorca, Spain Received 27 January 2003; accepted 11 July 2003

Abstract A simple indirect sequential injection spectrophotometric method for the determination of the UV-filter p-aminobenzoic acid (PABA) in sunscreen formulations is proposed. The determination is based on the reaction of PABA with hypochlorite in acidic medium and the subsequent measurement of the residual chlorine by using the well-known reaction with otolidine. The experimental variables involved in the sequential injection analysis (SIA) system (sandwich arrangement, volumes of sample and reagents, propulsion flow rate, reaction coil length) and adequate concentrations of reagents were studied. The SIA method has a linear range up to 20 ␮g ml−1 (3sy/x /b detection limit) of 1.0 ␮g ml−1 and a measurement throughput of 55 injections h−1 . Sunscreen cosmetics containing PABA were analyzed by the proposed method and the results were validated by comparison with those obtained by a liquid chromatographic procedure used as reference. The analytical features of the proposed method make it suitable for quality control of final products. © 2003 Elsevier B.V. All rights reserved. Keywords: p-Aminobenzoic acid (PABA); Sunscreen; Cosmetic; Sequential injection analysis (SIA); o-Tolidine; Hypochlorite

1. Introduction The use of sunscreen products helps to prevent or minimize the harmful effects that UV radiation from sunlight could cause on human health [1]. The large molar asborptivity of p-aminobenzoic acid (PABA) in the UVB region has made this compound suitable to be used as a UV filter in sunscreen formulations in order to avoid UV damage to the skin of sunbathing people. Consequently, PABA has been ∗ Corresponding author. Tel.: +34-96-3543175; fax: +34-96-3544436. E-mail address: [email protected] (A. Salvador).

the most commonly used UV filter of the post-World War II era [2,3]. However, nowadays many cosmetic manufacturers have been gradually phasing out its use in view of dermatological side effects observed, such as contact or photocontact dermatitis [2–5]. Moreover, different studies have shown that DNA is damaged after UV irradiation in the presence of PABA [6]. For these reasons, PABA has been gradually replaced by PABA esters like octyl dimethyl PABA. Nevertheless, the use of PABA is still allowed at 15% in EEUU [7] and 5% in Europe [8]. Since there are no official methods to determine PABA in sunscreen products, the development of adequate control analytical method is required.

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00873-0

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Techniques such as liquid chromatography (LC) [9–13], micellar electrokinetic capillary chromatography [13] and Raman spectrometry [14] have been employed for PABA determination in sunscreen products. No flow methods have been reported for PABA determination in sunscreens to date, except for our recent paper which is based on the use of a Bratton–Marshall type reaction [15]. Principles and application of sequential injection analysis (SIA) [16] methods in analytical chemistry have recently been reviewed [17–20]. SIA provides low reagent consumption, thus, generating low waste, which allows the toxicity and cost of the analytical procedure to be decreased [20]. SIA systems have been previously developed by our group to determine other UV filters [21–23]. The aim of this work is the development and validation of a SIA method for the determination of PABA in sunscreen formulations. The indirect spectrophotometric method proposed herein is based on the reaction between PABA and hypochlorite in acidic medium and the subsequent measurement of the residual chlorine by using the well-known reaction with o-tolidine. The o-tolidine–chlorine reaction has been extensively applied for residual chlorine determination in water using batch and flow injection analysis (FIA) strategies [24,25]. However, only March et al. [26] have used this reaction in a SIA system. Few articles involving the use of this reaction for indirect analysis have been found in the literature, such as those referring to hydrogen peroxide determination carried out

by Basfar and Demirata [27] and cyanide and thiocyanate determination by Apak et al. [28]. The reaction between PABA and hypochlorite is proposed in this paper with analytical purposes for the first time. The proposed indirect spectrophotometric method enables a sensitive and rapid online determination of PABA without prior treatment of samples, by using a SIA system.

2. Experimental 2.1. Apparatus The SIA system depicted in Fig. 1 was constructed with the following components: a Crison 2031 autoburette (Alella, Barcelona, Spain) equipped with a 5 ml syringe and an eight-channel Crison 2030 automatic valve (Alella) connected to a personal computer via an RS 232 C interface and controlled by home made software. A 10 mm QS 1000 (Hellma, Müllheim/Baden, Germany) flow-through cell (i.v. 18 ␮l) and PTFE tubing (0.5 mm i.d.) were also used. The measurements were carried out with a 8453 Hewlett-Packard UV-visible diode array spectrophotometer. A Hitachi LC apparatus, equipped with a Hitachi L-7100 high-pressure pump and a Hitachi L-7420 UV-Vis detector, was employed to carry out the determinations by the method used as reference. A LiChrospher® RP-18 (12.5 cm length ×

Fig. 1. The sequential injection analysis manifold. (1) carrier: water; (2) autoburette with 5 ml syringe; (3) holding coil (2 m); (4) eight-channel selector valve; (5) sample or standard solution in EtOH:1.0 M HCl = 30:70; (6) hypochlorite solution; (7) water; (8) o-tolidine in 1.0 M HCl; (9) reaction coil; (10) flow cell.

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4 mm i.d., 5 ␮m particle size, Merck) column was used. 2.2. Reagents PABA >99% from Guinama (Valencia, Spain) was used to prepare the standards. o-Tolidine (3,3 -dimethylbenzidine) from Aldrich (Barcelona), hydrochloric acid 37% (m/m) and a sodium hypochlorite 5% (m/v) solution from Scharlab (Barcelona) of analytical-reagent grade, ethanol 96% (v/v) cosmetic grade from Guinama and de-ionized water were used in the proposed method. EtOH absolute LC grade from Scharlab, acetic acid (HAc) of analytical-reagent grade from Panreac (Barcelona) and de-ionized water using a NANOpure II ultrapure water system from Barnstead (Boston, MA) were used as solvents in the reference method. Other reagents used to prepare laboratory-made sunscreens were of cosmetic-reagent grade and were purchased from Guinama. Sodium hypochlorite was titrated before use by employing a sodium thiosulphate solution previously standardized against potassium dichromate. The hypochlorite working solutions were prepared by dilution with water. 2.3. Samples Two commerical sunscreen samples (a lotion and a sun milk) containing PABA and other UV filters from Fortbenton Co. Laboratories (Buenos Aires, Argentina) were purchased at a local market. Three sunscreen sample containing known concentrations of PABA were prepared in our laboratory according to a habitual procedure followed in the cosmetic industries (provided by Guinama). These formulations also contained the usual ingredients employed in sunscreen formulations, such as a base cream with myristyl myristate, cetyl alcohol, glyceryl laurate, cetearyl octanoate, isopropyl myristate and other lipophilic components, avocado oil, dimethicone, Vitamin E, propylene glycol, hydroviton (an aqueous preparation containing amino acids, sodium lactate, urea, sodium chloride, and other hyrophilic compounds), parabenes and phenoxyethanol.

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2.4. Procedures 2.4.1. Reference method There are no official analytical methods for the determination of PABA. A LC method previously developed to determine other UV filters was used as a reference method [29]. An amount of 0.01–0.06 g of sample were dissolved and diluted to 25 ml with the mobile phase. The solution was filtered through Whatman 42 filter paper, and subsequently through a 0.45 ␮m nylon filter syringe before injection. Three replicates of each sunscreen were made. PABA standards (20–100 ␮g ml−1 ) were prepared in the same solvent as that of the samples and used for constructing the calibration graph. Twenty microlitres of standard and sample solutions were injected into the LC system and eluted by using EtOH:HAc:H2 O = 70:0.5:29.5 (v/v/v) as mobile phase, at a flow rate of 0.5 ml min−1 . The detection was performed by measuring the absorbance at 311 nm. 2.4.2. Proposed method: indirect SIA spectrophotometric determination An amount of 0.02–0.2 g of sunscreen was dissolved in and diluted to 25 ml with EtOH. An aliquot of 7.5 ml (previously filtered when containing titanium dioxide) was transferred to a 25 ml volumetric flask and made up to 25 ml with 1.0 M HCl. Three replicates of each sunscreen were made. PABA standard solutions (0–20 ␮g ml−1 ) in EtOH:1.0 M HCl = 30:70 (v/v) prepared from a 125 ␮g ml−1 PABA ethanolic stock solution, were used to construct the calibration graph. All samples (previously filtered through a 0.45 ␮m nylon filter syringe whenever required) and standard solutions were aspirated in triplicate into the SIA system depicted in Fig. 1. The measurements were carried out at 434 nm and corrected at 700 nm to minimize the effect of the refractive index variations. The aspiration and propulsion flow rates were 37.5 and 3.75 ml min−1 , respectively. A reaction coil length of 75 cm was employed. The following analytical cycle was used: 1. aspiration of 1000 ␮l of carrier (water); 2. aspiration of 50 ␮l of sample or standard solution; 3. aspiration of 50 ␮l of hypochlorite solution;

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4. aspiration of 60 ␮l of water; 5. aspiration of 80 ␮l of o-tolidine solution; 6. propulsion of 1240 ␮l to the detector through the reaction coil.

Initially, LC grade ethanol was used but the results obtained were comparable to those obtained when cosmetic grade ethanol was used. Thus, cosmetic grade ethanol was employed for further investigations owing to its lower cost.

3. Results and discussion

3.2.2. Study of experimental variables Previous experimental assays showed that solutions of 2.0 × 10−4 M hypochlorite in water and 2.0 × 10−4 M o-tolidine in 1.0 M HCl provided an adequate signal when they were used as reagents in the PABA determination by employing the SIA system described previously. Therefore, these concentrations were finally chosen. The concentration of PABA used to study the variables was 10 ␮g ml−1 , unless otherwise stated. The initial volume of hypochlorite, o-tolidine and PABA solutions and water tested was 50 ␮l in each case. A 3.75 ml min−1 flow rate and a coil length of 75 cm were used as initial conditions. The aspirated flow rate (37.5 ml min−1 ) was kept constant for technical reasons. To study the experimental variables, the absorbance signal from a PABA solution in EtOH:1.0 M HCl = 30:70 (v/v) solution was measured and the difference from a blank solution was measured. All the solutions were aspirated in triplicate. The studied variables were: sandwich arrangement, aspirated volumes of hypochlorite, o-tolidine, water and sample solutions together with the reaction coil length, and propulsion flow rate. Different sandwich arrangements were tested to ensure adequate sensitivity and precision. As the determination was carried out in two steps (reaction of PABA with hypochlorite in an acidic medium and the subsequent reaction of the remaining chlorine with o-tolidine), 50 ␮l of water was aspirated between the hypochlorite and o-tolidine solutions in order to prevent the occurrence of the reaction between the former compounds prior to the reaction between hypochlorite and PABA. The tested sequences were: PABA–ClO− –water–o-tolidine, ClO− –PABA–water–o-tolidine, ClO− –PABA–ClO− – water–o-tolidine and PABA–ClO− –PABA–water–otolidine. The blank solution was also aspirated instead of the PABA solution in order to compare the absorbance signals. Although, the reaction took place in all cases, the second sequence was rejected due to the low reproducibility attained. The remaining sequences

3.1. Reference method The linear equation obtained from the calibration graph was: APABA = (31 ± 4) × 104 + (155 ± 1) × 103 C (r2 = 0.9998; N = 5, C is expressed as ␮g ml−1 ) The results obtained by the LC method for the two commercial and the three laboratory-made sunscreen samples studied are shown in Table 1. 3.2. Proposed method 3.2.1. Previous studies Previous batch studies showed that pure ethanol destroys the yellow product formed by the reaction between o-tolidine and chlorine. This effect decreased, when the percentage of ethanol used as solvent diminished. A 30% (v/v) ethanol solution was chosen since it provided both good solubility of samples and adequate stability of the measuring compound. Thus, taking into account that an acidic medium is necessary to carry out the reaction, EtOH:1.0 M HCl = 30:70 (v/v) was finally chosen for sample and standard solutions. Table 1 Results obtained for PABA determination in sunscreens Samplea

A B C D E a

PABA concentrationb (%) Real content

Reference method

5.0 0.5 1.0 3.0 5.0

5.4 0.43 0.96 2.80 4.84

± ± ± ± ±

0.4 0.02 0.03 0.04 0.07

Proposed method 5.1 0.53 0.98 2.9 4.9

± ± ± ± ±

0.2 0.03 0.07 0.1 0.2

Samples C, D and E were laboratory-made samples. Average value from three replicate anaslysis ± standard deviation. b

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provided similar results and the simplest sequence PABA–ClO− –water–o-tolidine was finally chosen. The influence of the aspirated reagent volume was also studied. Volumes ranging from 25 to 100 ␮l for hypochlorite solution and 25 to 120 ␮l for o-tolidine solution were tested, as is shown in Figs. 2a and b. The highest sensitivity was obtained when 50 ␮l of hypochlorite solution and 80 ␮l of o-tolidine solution were used, and for this reason these values were selected in the subsequent variables studies. In order to prevent the reaction between both reagents (hypochlorite and o-tolidine) from taking place prior to the reaction between hypochlorite and PABA, a volume of water was aspirated between the hypochlorite and o-tolidine. The influence of this aspirated water volume was also studied. For this purpose, a solution of 2.2 × 10−4 M PABA (slightly more concentrated than hypochlorite), was aspirated. An excess of water hindered the reaction between hypochlorite and o-tolidine, thus, a volume of 60 ␮l of water was chosen (Fig. 2c). This value was used to carry out further studies. The aspirated sample volume was studied over the range 25–100 ␮l, as shown in Fig. 2d. The highest sensitivity was obtained for an aspiration volume of 50 ␮l and, therefore, was selected. The influence of the reaction coil length was investigated between 50 and 150 cm (see Fig. 2e). Although the highest sensitivity was achieved with a length of 50 cm, it was finally rejected due to the deformed peaks produced. Thus, 75 cm was used. The propulsion flow rate was tested over the range 3.75–12.5 ml min−1 . This parameter does not have a great influence on the signal, as shown in Fig. 2f. A value of 3.75 ml min−1 provided adequate sensitivity and precision. 3.2.3. Interference study The influence of other UV filters which could be mixed with PABA in sunscreen formulations was studied by aspirating 50 ␮l of a 10 ␮g ml−1 PABA solution to which interfering substances had previously been added. The tolerable interfering agent/analyte concentration ratios which provide changes in analyte signals <10% are listed in Table 2. As can be seen, the majority of the UV filters do not produce changes in the PABA signal at ratios lower than 30, except octyl dimethyl PABA

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and benzophenones, which could produce interferences at concentrations closer to that of the analyte. Since octyl dimethyl PABA has a similar absorption spectrum to PABA, they are not used as mixtures in sunscreens, therefore special attention has to be paid only if PABA is mixed with benzophenones. 3.2.4. Analytical figures of merit The calibration graph was non-linear and the trend was better described using logarithm of the absorbance signal. The obtained equation was: ln A434 = (0.29 ± 0.04) − (0.132 ± 0.003)C; r 2 = 0.9990; N = 5 (linear range 0–20 ␮g ml−1 ), where C is expressed as ␮g ml−1 . The limit of detection (LOD) estimated as 3sy/x /b, where sy/x is the standard deviation of the calibration graph and b the slope, was 1.0 ␮g ml−1 . The recovery of the method was studied by spiking ethanolic solutions of two commercial samples. The recoveries obtained were 99 and 102% for samples A and B, respectively, revealing the absence of any matrix effect. In order to evaluate the accuracy of the method, two commercial and three laboratory-made sunscreen samples were analyzed by the proposed SIA spectrophotometric method. The results (Table 1) were compared to both, the real contents and those obtained by a LC method (used as reference). A linear regression model led to the following equations: Y = (−0.01 ± 0.07) + (1.00 ± 0.02)X; r 2 = 0.9990; N=5

(compared with real contents)

Table 2 Tolerable interfering agent analyte concentration ratios for some potentially interfering compounds Interferent

Maximum tolerable concentration ratio

Octyl dimethyl PABA Benzophenone-3 Benzophenone-4 Octyl methoxycinnamate Homosalate Octyl salicylate Butyl methoxydibenzoylmethane

0.5 0.5 1.0 >30 >30 >30 >30

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Fig. 2. Effect of experimental variables affecting the analytical signal (mean value ± standard deviation of three measurements). Aspirated volumes: hypochlorite (a); o-tolidine (b); water (c); sample (d); reaction coil length (e); propulsion flow rate (f). Blank solution (䉬); PABA standard solution (䊏); signal difference (䉱).

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Y = (0.1 ± 0.1) + (0.96 ± 0.04)X; N=5

r 2 = 0.995;

(compared with the LC method)

where Y represents the results obtained by the proposed method and X are the real contents or the results obtained by the LC method used as reference, expressed as % PABA (m/m). The theoretical t-value [30] for a 95% confidence level and N − 2 degrees of freedom is 3.18 and the experimental t-values for the intercept and the slope were 0.15 and 0.18, respectively, when the results were compared with the real contents, and 0.94 and 1.13, respectively, when compared with the method used as reference, thus, proving the accuracy of the results. The R.S.D. of the concentration values obtained ranged from 3 to 7%. The measurement time of a solution was 65 s, and therefore the injection frequency is 55 h−1 . A complete cycle including three measurements per sample and a washing step took 275 s, which provided a sample throughput of 13 h−1 .

4. Conclusions A simple, rapid, accurate and precise SIA spectrophotometric method for p-aminobenzoic acid determination in sunscreens based on the reaction of this compound with hypochlorite in acidic medium and the subsequent measurement of residual chlorine with o-tolidine has been developed. The automation of the method by the use of a SIA system allows the amounts of reagents employed and thus, the waste generated to be decresed. The analytical features of the proposed method make it suitable for the quality control of final products.

Acknowledgements The authors acknowledge the financial support of the Spanish Ministry of Science and Technology for this work and the predoctoral grant given to A. Chisvert.

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