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Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection
Analytica Chimica Acta 588 (2007) 316–320
Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection Motoko Narita a , Kazuo Murakami a , Jean-Michel Kauffmann b,∗ a
Tokyo Kasei University, Faculty of Domestic Science, 1-18-1, Kaga, Itabashi, Tokyo 178-8602, Japan b Universit´ e Libre de Bruxelles, Pharmaceutical Institute, Campus Plaine, CP 205/6, Boulevard du Triomphe, 1050 Bruxelles, Belgium Received 7 September 2006; received in revised form 8 February 2007; accepted 13 February 2007 Available online 20 February 2007
Abstract The simultaneous determination of seven aminophenols, resorcinol and p-phenylenediamine in hair coloring products was performed by liquid chromatography (HPLC) with amperometric detection (ED). The aminophenols were separated on a ODS C18 reversed-phase column by isocratic elution with a mobile phase based on 0.1 M acetate buffer pH 4.5–methanol (90:10%, v/v) at a flow rate 0.8 mL min−1 . The limit of detection (S/N = 3) for the aminophenols was in the 15–40 pg (injected mass) range at an applied potential of 0.950 V versus Ag/AgCl. Peak heights for the aminophenols and the two others compounds were found to be linearly related to the amount injected, from 0.3 to 300 ng (r > 0.994–0.999).The relative standard deviation (R.S.D., n = 10) for 1 ng injected was comprised in the range from 2.5 to 6.2%, depending on the aminophenol tested. The present method minimizes troublesome and time-consuming pretreatment procedures and it was applied to the determination of aminophenols, resorcinol and phenylenediamine in hair coloring formulations. © 2007 Elsevier B.V. All rights reserved. Keywords: Aminophenol; Hair; Dye; Analysis; Liquid chromatography
1. Introduction Coal-tar dyes are used for hair coloring agent released in Japan. The dying formulations contain dye precursors such as aminophenol and p-phenylenediamine compounds. It is well reported that these dye products are of health concern due to risks of allergic dermatitis, nephrotoxic and carcinogenic effects [1,2]. Mixtures of hair coloring dyes are better analyzed using separation methods such as gas chromatography (GC), liquid chromatography (HPLC), and capillary electrophoresis (CE) [3–9]. Generally UV and mass spectrometric detectors are applied. Yet, electroanalytical methods are well suited for the determination of phenol and derivatives and especially aminophenols [10]. To the best of our knowledge amperometric detection coupled to LC has not yet been investigated for the sensitive detection and determination of aminophenols and dye precursors in hair dye solutions.
∗
Corresponding author. Tel.: +32 2 6505215; fax: +32 2 6505225. E-mail address: [email protected] (J.-M. Kauffmann).
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.02.023
In the present study, an LC–ED method was developed for the determination of 7 aminophenol derivatives and two other compounds namely: p-aminophenol (p-AP); p-methylaminophenol (p-MAP); m-aminophenol (m-AP); o-aminophenol (o-AP); 2-amino-4-nitrophenol (2-A-4-NP); 2-amino-5-nitrophenol (2A-5-NP), resorcinol, p-phenylenediamine and 5-amino-2methylphenol (5-A-2-MP) in hair coloring products released in Japan. 2. Experimental 2.1. Chemicals and reagents p-Aminophenol, o-aminophenol, m-aminophenol, 2-amino4-nitrophenol, p-methylaminophenol sulfate, 5-amino-2-methylphenol, resorcinol, and p-phenylenediamine were obtained from Wako (Osaka, Japan). 2-Amino-5-nitrophenol was obtained from Tokyo Kasei Industrial (Tokyo, Japan). The purity of the studied compounds was above 98% except for o-AP (97.0%). Methanol of HPLC grade, anhydrous sodium acetate and acetic acid were from Wako (Osaka, Japan). Other reagents
M. Narita et al. / Analytica Chimica Acta 588 (2007) 316–320
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used were of analytical grade. Water was from a Milli-Q water purification system Millipore (Bedford, MA, USA). 2.2. Apparatus The LC-ED system consisted of a pump Shodex DS-10 Showa Denko (Tokyo, Japan), an electrochemical detector LC4A, BAS (West Lafayette, USA) and a UV spectrophotometer detector Shimadzu (Kyoto, Japan). The commercially available electrochemical cell (BAS) was constructed from a glassy carbon working electrode, an Ag/AgCl reference electrode, and a stainless steel auxiliary electrode. With the spectrophotometer detector, all compounds were detected at 254 nm. The recording and integrating device was a SIC chromatocorder 21 SIC (Tokyo, Japan). The separation column was a Shodex C18M 4D (ODS 5 m), reversed-phase column (4.6 mm i.d. × 150 mm, Showa Denko (Tokyo, Japan). Sample was injected with a Rheodyne 7125 (Cotati, CA, USA) syringe loading injector (20 L loop). 2.3. Mobile phase The mobile phase was methanol (10 vol%)–0.1 M acetate buffer pH 4.5 (90 vol%) and was delivered at a flow rate of 0.8 mL min−1 .
Fig. 1. Effect of MeOH in mobile phase on the retention time of seven aminophenols and two other compounds (10 ng injected). (1) 2-A-5-NP; (2) 2-A-4-NP; (3) 5-A-2-MP; (4) o-AP; (5) resorcinol; (6) m-AP; (7) p-MAP; (8) p-AP; (9) p-phenylenediamine LC conditions are as in Section 2.
3. Results and discussions 3.1. Chromatographic separation
2.4. HPLC-ED conditions The stock solution of the investigated compounds was prepared in methanol. The standard solutions containing the aminophenols were injected into the LC system and the amperometric response recorded in the applied potential range from 0.1 to +1.2 V. The peak height was plotted versus the applied potential. The final working electrode potential was set at +0.95 V versus Ag/AgCl. The working electrode was smoothed on a polishing cloth, in the conventional way, once a week. 2.5. Preparation of the analyzed sample Fourteen kinds of hair coloring agents were obtained from three companies. Ten samples of coloring agents were obtained from A company, three kinds of them from B company and one from C company. The 14 kinds of hair coloring formulations displayed sample numbers and colors of dyed hairs as follows: Sample 1 dark chestnut, sample 2 natural chestnut, sample 3 natural black, sample 4 milk caramel, sample 5 mocha beige, sample 6 caramel orange, sample 7 honey brown, sample 8 natural black, sample 9 brown, sample 10 brown II from A company, sample 11 slightly bright chestnut, sample 12 chestnut, sample 13 slightly dark chestnut from B company and sample 14 natural black II from C company. A precisely weighed quantity (0.1 g) of hair coloring product was dissolved in 10ml of methanol and sonicated for 1 min. This solution was diluted 1000 times with methanol and filtered through a 0.45 m membrane filter. A volume of 10 L of the diluted solution was injected into the LC system.
The effect of methanol content, concentration of buffer solution and pH in mobile phase on elution of seven aminophenols, resorcinol and p-phenylenediamine likely present in hair coloring formulations. The data in Fig. 1 illustrate the influence of methanol content onto the elution for the seven aminophenols and two other compounds studied. With respect to pH effect, the peak height and retention time were not influenced by the pH of the acetate buffer when varied from 3.5 to 5.5. When the acetate buffer concentration varied from 0.05 to 0.3 M, the peak height and retention time were not influenced. The methanol–0.1 M acetate buffer pH 4.5 (10:90%, v/v) composition was finally selected as it gave the best separation of the studied amino phenols within a short analysis period of time (less than 25 min). The elution order for the nine compounds was pphenylenediamine (tr = 2.2min), p-AP (tr = 2.5 min), p-MAP (tr = 3.6 min), m-AP (tr = 4.8 min), resorcinol (tr = 6.0), o-AP (tr = 9.0 min), 5-amino-2-methylphenol (tr = 12.5), 2-A-4-NP (tr = 20.6 min) and 2-A-5-NP (tr = 24.0 min). Resolutions of p-phenylenediamine ∼ p-AP, p-AP ∼ p-MAP, p-MAP ∼ m-AP, m-AP ∼ resorcinol, resorcinol ∼ o-AP, o-AP ∼ 5-A-2-MP, 5-A2-MP ∼ 2-A-4-NP and 2-A-4-NP ∼ 2-A-5-NP were 0.6, 2.2, 3.0, 3.0, 7.5, 7.0, 11.6 and 4.3, respectively. 3.2. Applied potential In order to determine the potential allowing the detection of the aminophenols investigated, the hydrodynamic voltammogram was established with the above selected mobile phase. As illustrated in Fig. 2, the aminophenols were all oxidized at an applied potential lower than 1 V. Except for compounds 1
318
M. Narita et al. / Analytica Chimica Acta 588 (2007) 316–320 Table 1 Linear range, correlation coefficient and repeatability of aminophenols by HPLC-ED Compound
p-AP p-MAP m-AP o-AP 5-A-2-MP 2-A-4-NP 2-A-5-NP
Linear range ng 10 L−1
0.3–300 0.3–300 0.3–300 0.3–300 0.3–300 0.3–300 0.3–300
Corr. coeff. (r)
0.999 0.999 0.997 0.994 0.999 0.999 1
mdm
15 30 30 25 35 40 35
Repeatability (n = 10) Within-day
Inter-day
(a)
(b)
(c)
(d)
5.5 6.5 8.6 6.7 5.0 9.3 9.0
4.5 2.5 6.2 3.5 4.5 5.9 5.9
6.5 2.1 7.0 5.2 6.6 8.0 12.7
5.5 5.1 7.2 5.3 6.5 8.0 14.8
R.S.D. values (%): (a)100 pg, (b) 1 ng, (c) 50 ng and (d) 300 ng. mdm: minimum detectable mass (pg, 10 L−1 ).
Fig. 2. Hydrodynamic voltammograms of seven aminophenols and two other compounds (50 ng injected). (1) m-AP; (2) o-AP; (3) p-phenylenediamine; (4) 5-A-2-MP; (5) resorcinol; (6) p-AP; (7) 2-A-4-NP; (8) p-MAP; (9) 2-A-5-NP. HPLC conditions: column: ODS column (150 mm × 4.6 ID); column temperature, 40 ◦ C; mobile phase, methanol-acetate buffer solution (10:90, v/v); flow rate, 0.80 mL min−1 .
and 2, all gave only one well defined wave till +0.80 V with some plateau distortion above that potential attributed to surface phenomena (current decrease) or to a subsequent electrooxidation process (current increase). Compounds 1 and 2 gave a higher signal with a distorted plateau above 0.95 V. Since the background current raised by increasing the applied potential, the selected potential was set at +0.95 V versus Ag/AgCl, i.e., not too positive but high enough to detect all the investigated molecules in the diffusion controlled domain. Fig. 3 represents a typical chromatogram obtained under the selected experimental conditions.
3.3. Determination of aminophenols standard solutions The peak heights were linearly related to the concentration of the respective injected aminophenols in the standard mixture from 0.3 ng to 300 ng (10 L injected), the response leveled off at higher concentrations (likely attributed to surface effects such as fouling). Linear calibration curves were obtained. Analytical figures of merit are shown in Table 1. The limit of detection for the different investigated molecules (S/N = 3) were 15–40 pg 10 L−1 . The relative standard deviation on ten successive injections (within-day) was in the range 2.5–6.2% and 5.5–9.3% for 1 and 100 pg injected, respectively. The inter-day repeatability was in the range of 2.1–12.7% and 5.1–14.8% (p-phenylenediamine = 23.2%) for 50 ng and 300 ng injected, respectively. Aminophenol recoveries for spiked samples (50 ng aminophenol added to 1 g hair coloring agent) were in the 96.4–100.4% range and the R.S.D.s (n = 5) were in the range 2.6–5.5%. The good recoveries obtained were likely related to the fact that the hair coloring agent solution to be analyzed needed no complex treatment other than dilution with methanol. 3.4. Application to real samples Different hair coloring agents were tested for the presence of the investigated aminophenols. Table 2 lists the result obtained
Table 2 Recovery of aminophenols from similar hair coloring agent without dyes Compound
Fig. 3. Typical chromatogram of seven aminophenols and two other compounds (50 ng injected). (1) p-phenylenediamine; (2) p-AP; (3) p-MAP; (4) m-AP; (5) resorcinol; (6) o-AP; (7) 5-A-2-MP; (8) 2-A-4-NP; (9) 2-A-5-NP. Experimental conditions as in Fig. 2.
p-AP p-MAP m-AP o-AP 5-A-2-MP 2-A-4-NP 2-A-5-NP
Precision concentration (ng g−1 ) Nominal
Found (mean) (n = 5)
50 50 50 50 50 50 50
49.8 48.4 49.4 50.2 49.2 48.2 48.2
R.S.D. (%)
Recovery (%)
2.6 3.8 2.6 2.7 4.8 5.5 4.7
100 97 100 96 98 99 96
± ± ± ± ± ± ±
3 4 3 3 3 6 5
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Table 3 Aminophenols in hair coloring agents (%) Sample no.
Color
p-AP
p-MAP
m-AP
o-AP
5-A-2-MP
Company
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Profound chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly bright chestnut Chestnut Slightly dark chestnut Slightly dark chestnut
0.920 0.280 – 0.150 0.560 0.390 – 1.150 1.170 0.130 1.380 1.700 1.780 –
0.100 – – – – 0.020 – – – 0.030 – – – –
– 0.002 0.760 0.003 0.020 0.020 – 0.180 0.070 0.050 0.230 0.320 0.620 –
– 0.040 0.630 0.030 0.030 0.030 0.070 0.050 – 0.070 0.310 0.230 0.250 0.110
0.190 – – 0.140 1.020 0.010 – – 1.600 – 1.180 1.300 – 1.680
A A A A A A A A A B B B B C
with 13 kinds of hair coloring agents. Four aminophenols (p-AP, p-MAP, m-AP and o-AP) were detected in hair coloring agents using the developed LC-ED procedure (Table 3). Fig. 4 shows a representative chromatogram in a commercial hair coloring formulation. Three studied aminophenols (peaks 2, 3 and 5) and peaks 1, 4 and 6 were identified as p-phenylenediamine, resorcinol and 5-amino-2-methylphenol (5-A-2-MP), respectively. The peak identity was also confirmed by LC-UV. The peak corresponding to p-phenylenediamine overlapped with the solvent (methanol) peak. The relationship between dye chromatic properties and levels of aminophenols in hair coloring agents were compared with three kinds of agents produced by the same company (Fig. 5 for sample nos. 11–13). Aminophenol content tended to be high in hair coloring products recommended Fig. 5. Patterns of relationship between chromatic deepness (chestnut) and aminophenol content (sample nos. 11–13B company): (1) slightly dark chestnut, (2) chestnut, (3) slightly bright chestnut.
for white hairs. The content of p-AP and m-AP depended on chestnut deepness. 4. Conclusion The present study showed that aminophenols, phenols and phenylenediamine, used in hair coloring products can be readily determined in complex mixtures with high sensitivity by LC-ED. The isocratic chromatographic conditions required no special reagents, few quantity of sample are needed and the preparation of the solution for the assay was simple to implement since no tedious pretreatment steps were needed. References
Fig. 4. Chromatogram of aminophenols in a hair coloring agent (sample no. 11): (1) p-phenylenediamine; (2) p-AP; (3) m-AP; (4) resorcinol; (5) o-AP; (6) 5-A-2-MP. Experimental conditions as in Fig. 2.
[1] G.J. Nohynek, R. Fautz, F. Benech-Kieffer, H. Toutain, Food Chem. Toxicol. 42 (2004) 517. [2] G.J. Nohynek, F.D. Duche, A. Garrigues, P.-A. Meunier, H. Toutain, J. Leclaire, Toxicol. Lett. 158 (2005) 196. [3] C.-E. Lin, Y.-T. Chen, T.-Z. Wang, J. Chromatogr. A 837 (1999) 241.
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[4] S.-P. Wang, T.-H. Huang, Anal. Chim. Acta 534 (2005) 207. [5] U. Vincent, G. Bordin, A.R. Rodriguez, J. Cosmetic Sci. 53 (2002) 101. [6] B.H. Shao, X.Z. Xu, J.W. Yan, X.Y. Fu, J. Liq. Chromatogr. Rel. Technol. 24 (2001) 241.
[7] [8] [9] [10]
S.C. Rastogi, J. Sep. Sci. 24 (2001) 173. N.A. Penner, P.N. Nesterenko, Analyst 125 (2000) 1249. J. Zhou, H. Xu, G.-H. Wan, C.-F. Duan, H. Cui, Talanta 64 (2004) 467. N.S. Lawrence, E.L. Beckett, J. Davis, R.G. Compton, Analyst 126 (2001) 1897.
Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection Motoko Narita a , Kazuo Murakami a , Jean-Michel Kauffmann b,∗ a
Tokyo Kasei University, Faculty of Domestic Science, 1-18-1, Kaga, Itabashi, Tokyo 178-8602, Japan b Universit´ e Libre de Bruxelles, Pharmaceutical Institute, Campus Plaine, CP 205/6, Boulevard du Triomphe, 1050 Bruxelles, Belgium Received 7 September 2006; received in revised form 8 February 2007; accepted 13 February 2007 Available online 20 February 2007
Abstract The simultaneous determination of seven aminophenols, resorcinol and p-phenylenediamine in hair coloring products was performed by liquid chromatography (HPLC) with amperometric detection (ED). The aminophenols were separated on a ODS C18 reversed-phase column by isocratic elution with a mobile phase based on 0.1 M acetate buffer pH 4.5–methanol (90:10%, v/v) at a flow rate 0.8 mL min−1 . The limit of detection (S/N = 3) for the aminophenols was in the 15–40 pg (injected mass) range at an applied potential of 0.950 V versus Ag/AgCl. Peak heights for the aminophenols and the two others compounds were found to be linearly related to the amount injected, from 0.3 to 300 ng (r > 0.994–0.999).The relative standard deviation (R.S.D., n = 10) for 1 ng injected was comprised in the range from 2.5 to 6.2%, depending on the aminophenol tested. The present method minimizes troublesome and time-consuming pretreatment procedures and it was applied to the determination of aminophenols, resorcinol and phenylenediamine in hair coloring formulations. © 2007 Elsevier B.V. All rights reserved. Keywords: Aminophenol; Hair; Dye; Analysis; Liquid chromatography
1. Introduction Coal-tar dyes are used for hair coloring agent released in Japan. The dying formulations contain dye precursors such as aminophenol and p-phenylenediamine compounds. It is well reported that these dye products are of health concern due to risks of allergic dermatitis, nephrotoxic and carcinogenic effects [1,2]. Mixtures of hair coloring dyes are better analyzed using separation methods such as gas chromatography (GC), liquid chromatography (HPLC), and capillary electrophoresis (CE) [3–9]. Generally UV and mass spectrometric detectors are applied. Yet, electroanalytical methods are well suited for the determination of phenol and derivatives and especially aminophenols [10]. To the best of our knowledge amperometric detection coupled to LC has not yet been investigated for the sensitive detection and determination of aminophenols and dye precursors in hair dye solutions.
∗
Corresponding author. Tel.: +32 2 6505215; fax: +32 2 6505225. E-mail address: [email protected] (J.-M. Kauffmann).
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.02.023
In the present study, an LC–ED method was developed for the determination of 7 aminophenol derivatives and two other compounds namely: p-aminophenol (p-AP); p-methylaminophenol (p-MAP); m-aminophenol (m-AP); o-aminophenol (o-AP); 2-amino-4-nitrophenol (2-A-4-NP); 2-amino-5-nitrophenol (2A-5-NP), resorcinol, p-phenylenediamine and 5-amino-2methylphenol (5-A-2-MP) in hair coloring products released in Japan. 2. Experimental 2.1. Chemicals and reagents p-Aminophenol, o-aminophenol, m-aminophenol, 2-amino4-nitrophenol, p-methylaminophenol sulfate, 5-amino-2-methylphenol, resorcinol, and p-phenylenediamine were obtained from Wako (Osaka, Japan). 2-Amino-5-nitrophenol was obtained from Tokyo Kasei Industrial (Tokyo, Japan). The purity of the studied compounds was above 98% except for o-AP (97.0%). Methanol of HPLC grade, anhydrous sodium acetate and acetic acid were from Wako (Osaka, Japan). Other reagents
M. Narita et al. / Analytica Chimica Acta 588 (2007) 316–320
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used were of analytical grade. Water was from a Milli-Q water purification system Millipore (Bedford, MA, USA). 2.2. Apparatus The LC-ED system consisted of a pump Shodex DS-10 Showa Denko (Tokyo, Japan), an electrochemical detector LC4A, BAS (West Lafayette, USA) and a UV spectrophotometer detector Shimadzu (Kyoto, Japan). The commercially available electrochemical cell (BAS) was constructed from a glassy carbon working electrode, an Ag/AgCl reference electrode, and a stainless steel auxiliary electrode. With the spectrophotometer detector, all compounds were detected at 254 nm. The recording and integrating device was a SIC chromatocorder 21 SIC (Tokyo, Japan). The separation column was a Shodex C18M 4D (ODS 5 m), reversed-phase column (4.6 mm i.d. × 150 mm, Showa Denko (Tokyo, Japan). Sample was injected with a Rheodyne 7125 (Cotati, CA, USA) syringe loading injector (20 L loop). 2.3. Mobile phase The mobile phase was methanol (10 vol%)–0.1 M acetate buffer pH 4.5 (90 vol%) and was delivered at a flow rate of 0.8 mL min−1 .
Fig. 1. Effect of MeOH in mobile phase on the retention time of seven aminophenols and two other compounds (10 ng injected). (1) 2-A-5-NP; (2) 2-A-4-NP; (3) 5-A-2-MP; (4) o-AP; (5) resorcinol; (6) m-AP; (7) p-MAP; (8) p-AP; (9) p-phenylenediamine LC conditions are as in Section 2.
3. Results and discussions 3.1. Chromatographic separation
2.4. HPLC-ED conditions The stock solution of the investigated compounds was prepared in methanol. The standard solutions containing the aminophenols were injected into the LC system and the amperometric response recorded in the applied potential range from 0.1 to +1.2 V. The peak height was plotted versus the applied potential. The final working electrode potential was set at +0.95 V versus Ag/AgCl. The working electrode was smoothed on a polishing cloth, in the conventional way, once a week. 2.5. Preparation of the analyzed sample Fourteen kinds of hair coloring agents were obtained from three companies. Ten samples of coloring agents were obtained from A company, three kinds of them from B company and one from C company. The 14 kinds of hair coloring formulations displayed sample numbers and colors of dyed hairs as follows: Sample 1 dark chestnut, sample 2 natural chestnut, sample 3 natural black, sample 4 milk caramel, sample 5 mocha beige, sample 6 caramel orange, sample 7 honey brown, sample 8 natural black, sample 9 brown, sample 10 brown II from A company, sample 11 slightly bright chestnut, sample 12 chestnut, sample 13 slightly dark chestnut from B company and sample 14 natural black II from C company. A precisely weighed quantity (0.1 g) of hair coloring product was dissolved in 10ml of methanol and sonicated for 1 min. This solution was diluted 1000 times with methanol and filtered through a 0.45 m membrane filter. A volume of 10 L of the diluted solution was injected into the LC system.
The effect of methanol content, concentration of buffer solution and pH in mobile phase on elution of seven aminophenols, resorcinol and p-phenylenediamine likely present in hair coloring formulations. The data in Fig. 1 illustrate the influence of methanol content onto the elution for the seven aminophenols and two other compounds studied. With respect to pH effect, the peak height and retention time were not influenced by the pH of the acetate buffer when varied from 3.5 to 5.5. When the acetate buffer concentration varied from 0.05 to 0.3 M, the peak height and retention time were not influenced. The methanol–0.1 M acetate buffer pH 4.5 (10:90%, v/v) composition was finally selected as it gave the best separation of the studied amino phenols within a short analysis period of time (less than 25 min). The elution order for the nine compounds was pphenylenediamine (tr = 2.2min), p-AP (tr = 2.5 min), p-MAP (tr = 3.6 min), m-AP (tr = 4.8 min), resorcinol (tr = 6.0), o-AP (tr = 9.0 min), 5-amino-2-methylphenol (tr = 12.5), 2-A-4-NP (tr = 20.6 min) and 2-A-5-NP (tr = 24.0 min). Resolutions of p-phenylenediamine ∼ p-AP, p-AP ∼ p-MAP, p-MAP ∼ m-AP, m-AP ∼ resorcinol, resorcinol ∼ o-AP, o-AP ∼ 5-A-2-MP, 5-A2-MP ∼ 2-A-4-NP and 2-A-4-NP ∼ 2-A-5-NP were 0.6, 2.2, 3.0, 3.0, 7.5, 7.0, 11.6 and 4.3, respectively. 3.2. Applied potential In order to determine the potential allowing the detection of the aminophenols investigated, the hydrodynamic voltammogram was established with the above selected mobile phase. As illustrated in Fig. 2, the aminophenols were all oxidized at an applied potential lower than 1 V. Except for compounds 1
318
M. Narita et al. / Analytica Chimica Acta 588 (2007) 316–320 Table 1 Linear range, correlation coefficient and repeatability of aminophenols by HPLC-ED Compound
p-AP p-MAP m-AP o-AP 5-A-2-MP 2-A-4-NP 2-A-5-NP
Linear range ng 10 L−1
0.3–300 0.3–300 0.3–300 0.3–300 0.3–300 0.3–300 0.3–300
Corr. coeff. (r)
0.999 0.999 0.997 0.994 0.999 0.999 1
mdm
15 30 30 25 35 40 35
Repeatability (n = 10) Within-day
Inter-day
(a)
(b)
(c)
(d)
5.5 6.5 8.6 6.7 5.0 9.3 9.0
4.5 2.5 6.2 3.5 4.5 5.9 5.9
6.5 2.1 7.0 5.2 6.6 8.0 12.7
5.5 5.1 7.2 5.3 6.5 8.0 14.8
R.S.D. values (%): (a)100 pg, (b) 1 ng, (c) 50 ng and (d) 300 ng. mdm: minimum detectable mass (pg, 10 L−1 ).
Fig. 2. Hydrodynamic voltammograms of seven aminophenols and two other compounds (50 ng injected). (1) m-AP; (2) o-AP; (3) p-phenylenediamine; (4) 5-A-2-MP; (5) resorcinol; (6) p-AP; (7) 2-A-4-NP; (8) p-MAP; (9) 2-A-5-NP. HPLC conditions: column: ODS column (150 mm × 4.6 ID); column temperature, 40 ◦ C; mobile phase, methanol-acetate buffer solution (10:90, v/v); flow rate, 0.80 mL min−1 .
and 2, all gave only one well defined wave till +0.80 V with some plateau distortion above that potential attributed to surface phenomena (current decrease) or to a subsequent electrooxidation process (current increase). Compounds 1 and 2 gave a higher signal with a distorted plateau above 0.95 V. Since the background current raised by increasing the applied potential, the selected potential was set at +0.95 V versus Ag/AgCl, i.e., not too positive but high enough to detect all the investigated molecules in the diffusion controlled domain. Fig. 3 represents a typical chromatogram obtained under the selected experimental conditions.
3.3. Determination of aminophenols standard solutions The peak heights were linearly related to the concentration of the respective injected aminophenols in the standard mixture from 0.3 ng to 300 ng (10 L injected), the response leveled off at higher concentrations (likely attributed to surface effects such as fouling). Linear calibration curves were obtained. Analytical figures of merit are shown in Table 1. The limit of detection for the different investigated molecules (S/N = 3) were 15–40 pg 10 L−1 . The relative standard deviation on ten successive injections (within-day) was in the range 2.5–6.2% and 5.5–9.3% for 1 and 100 pg injected, respectively. The inter-day repeatability was in the range of 2.1–12.7% and 5.1–14.8% (p-phenylenediamine = 23.2%) for 50 ng and 300 ng injected, respectively. Aminophenol recoveries for spiked samples (50 ng aminophenol added to 1 g hair coloring agent) were in the 96.4–100.4% range and the R.S.D.s (n = 5) were in the range 2.6–5.5%. The good recoveries obtained were likely related to the fact that the hair coloring agent solution to be analyzed needed no complex treatment other than dilution with methanol. 3.4. Application to real samples Different hair coloring agents were tested for the presence of the investigated aminophenols. Table 2 lists the result obtained
Table 2 Recovery of aminophenols from similar hair coloring agent without dyes Compound
Fig. 3. Typical chromatogram of seven aminophenols and two other compounds (50 ng injected). (1) p-phenylenediamine; (2) p-AP; (3) p-MAP; (4) m-AP; (5) resorcinol; (6) o-AP; (7) 5-A-2-MP; (8) 2-A-4-NP; (9) 2-A-5-NP. Experimental conditions as in Fig. 2.
p-AP p-MAP m-AP o-AP 5-A-2-MP 2-A-4-NP 2-A-5-NP
Precision concentration (ng g−1 ) Nominal
Found (mean) (n = 5)
50 50 50 50 50 50 50
49.8 48.4 49.4 50.2 49.2 48.2 48.2
R.S.D. (%)
Recovery (%)
2.6 3.8 2.6 2.7 4.8 5.5 4.7
100 97 100 96 98 99 96
± ± ± ± ± ± ±
3 4 3 3 3 6 5
M. Narita et al. / Analytica Chimica Acta 588 (2007) 316–320
319
Table 3 Aminophenols in hair coloring agents (%) Sample no.
Color
p-AP
p-MAP
m-AP
o-AP
5-A-2-MP
Company
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Profound chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly dark chestnut Slightly bright chestnut Chestnut Slightly dark chestnut Slightly dark chestnut
0.920 0.280 – 0.150 0.560 0.390 – 1.150 1.170 0.130 1.380 1.700 1.780 –
0.100 – – – – 0.020 – – – 0.030 – – – –
– 0.002 0.760 0.003 0.020 0.020 – 0.180 0.070 0.050 0.230 0.320 0.620 –
– 0.040 0.630 0.030 0.030 0.030 0.070 0.050 – 0.070 0.310 0.230 0.250 0.110
0.190 – – 0.140 1.020 0.010 – – 1.600 – 1.180 1.300 – 1.680
A A A A A A A A A B B B B C
with 13 kinds of hair coloring agents. Four aminophenols (p-AP, p-MAP, m-AP and o-AP) were detected in hair coloring agents using the developed LC-ED procedure (Table 3). Fig. 4 shows a representative chromatogram in a commercial hair coloring formulation. Three studied aminophenols (peaks 2, 3 and 5) and peaks 1, 4 and 6 were identified as p-phenylenediamine, resorcinol and 5-amino-2-methylphenol (5-A-2-MP), respectively. The peak identity was also confirmed by LC-UV. The peak corresponding to p-phenylenediamine overlapped with the solvent (methanol) peak. The relationship between dye chromatic properties and levels of aminophenols in hair coloring agents were compared with three kinds of agents produced by the same company (Fig. 5 for sample nos. 11–13). Aminophenol content tended to be high in hair coloring products recommended Fig. 5. Patterns of relationship between chromatic deepness (chestnut) and aminophenol content (sample nos. 11–13B company): (1) slightly dark chestnut, (2) chestnut, (3) slightly bright chestnut.
for white hairs. The content of p-AP and m-AP depended on chestnut deepness. 4. Conclusion The present study showed that aminophenols, phenols and phenylenediamine, used in hair coloring products can be readily determined in complex mixtures with high sensitivity by LC-ED. The isocratic chromatographic conditions required no special reagents, few quantity of sample are needed and the preparation of the solution for the assay was simple to implement since no tedious pretreatment steps were needed. References
Fig. 4. Chromatogram of aminophenols in a hair coloring agent (sample no. 11): (1) p-phenylenediamine; (2) p-AP; (3) m-AP; (4) resorcinol; (5) o-AP; (6) 5-A-2-MP. Experimental conditions as in Fig. 2.
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