International Journal of Cosmetic Science, 2015, 37, 306–311
doi: 10.1111/ics.12200
Studies on the stability of preservatives under subcritical water conditions B. Kapalavavi*, R. Marple†, C. Gamsky† and Y. Yang* *Department of Chemistry, East Carolina University, Greenville, NC 27858, and †Global Analytical Capability Organization, The Procter & Gamble Company, Cincinnati, OH 45241, U.S.A.
Received 14 November 2014, Accepted 21 December 2014
Keywords: benzyl alcohol, degradation, high-performance liquid chromatography, parabens, preservatives, skincare creams, stability, subcritical water chromatography
Synopsis OBJECTIVE: The goal of this work was to further validate the subcritical water chromatography (SBWC) methods for separation and analysis of preservatives through the evaluation of analyte stability in subcritical water. METHODS: In this study, the degradation of preservatives was investigated at temperatures of 100–200°C using two different approaches. First, the peak areas obtained by SBWC at high temperatures were compared with those achieved using the traditional high-performance liquid chromatography (HPLC) at 25°C. In the second approach, several preservatives and water were loaded into a vessel and heated at high temperatures for 30 or 60 min. The heated mixtures were then analysed by GC/MS to determine the stability of preservatives. RESULTS: The t- and F-test on the results of the first approach reveal that the peak areas achieved by HPLC and SBWC are not significantly different at the 95% confidence level, meaning that the preservatives studied are stable during the high-temperature SBWC runs. Although the results of the second approach show approximately 10% degradation of preservatives into mainly p-hydroxybenzoic acid and phenol at 200°C, the preservatives studied are stable at 100 and 150°C. This is in good agreement with the validation results obtained by the first approach. CONCLUSION: The findings of this work confirm that SBWC methods at temperatures up to 150°C are reliable for separation and analysis of preservatives in cosmetic and other samples. sume Re OBJECTIF: Le but de ce travail est de valider davantage les methodes de chromatographies d’eau sous-critique (SBWC) pour la separation et l’analyse des conservateurs, par l’evaluation de la stabilite de l’analyte dans l’eau sous-critique. METHODES: Dans cette etude, la degradation des agents de con des temperatures de 100 a 200°C en utiliservation a ete etudiee a sant deux approches differentes. Premierement, les surfaces des pics obtenus par SBWC a des temperatures elevees ont ete comparees 25°C. avec celles obtenues en utilisant la HPLC traditionnelle a Dans la seconde approche, plusieurs agents de conservation et de Correspondence: Yu Yang, Department of Chemistry, East Carolina University, Greenville, NC 27858, U.S.A. Tel.: 1-252-328-9811; fax: 1-252-328-6210; e-mail: [email protected]
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des temperatures l’eau ont ete charges dans une cuve et chauffes a elevees pendant 30 ou 60 min. Les melanges chauffes ont ensuite ete analyses par GC/MS pour determiner la stabilite de conservation. RESULTATS: Les tests t et F sur les resultats de la premiere methode montrent que les surfaces des pics obtenus par HPLC et SBWC ne sont pas significativement differentes au seuil de confiance de 95%, ce qui signifie que les agents conservateurs etudies sont stables pendant l’execution de la SBWC a haute temperature. Bien que les resultats de la seconde approche montrent environ 10% de degradation des conservateurs vers principalement de l’acide p-hydroxybenzo€ıque et de phenol a 200°C, les conservateurs etudies sont stables entre 100 et 150°C. Ceci est en bon accord avec les resultats de la validation obtenus par la premiere methode. CONCLUSION: Les resultats de ces travaux confirment que les des temperamethodes de chromatographie de l’eau subcritique a tures allant jusqu’ a 150°C sont fiables pour la separation et l’analyse des conservateurs dans les cosmetiques et d’autres echantillons. Introduction Preservatives are used in over 22 000 cosmetic products with concentration of up to 0.4% for a single paraben. Obviously, preservative concentrations in cosmetic products must be accurately determined before the products can be released. The most common technique for the analysis of preservatives is high-performance liquid chromatography (HPLC). However, HPLC requires a huge amount of organic solvents for its mobile phase. These organic solvents used in HPLC mobile phase are not only hazardous and expensive in purchasing, but also require waste disposal that further increases the costs. Fortunately, pure water at elevated temperatures can be used as the mobile phase to achieve reversedphase separations [1–9]. We have recently developed subcritical water chromatography (SBWC) methods for separation and analysis of niacinamide, sunscreens and preservatives in skincare products [4–6]. Our work demonstrated that SBWC can achieve the same analytical quality as that obtained by HPLC, thus eliminating the hazardous and expensive organic solvents required in HPLC. The temperature required in SBWC separations depends on the polarity of analytes as well as the property of the stationary phase. Please note that SBWC is a technique of reversed-phase liquid chromatography where the mobile phase is more polar than the stationary phase. In general, less polar solutes require less polar
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
B. Kapalavavi et al.
Stability of preservatives in subcritical water
mobile phase to be efficiently eluted. This in turn requires higher temperature that decreases the polarity of subcritical water used as the mobile phase in SBWC. However, the high temperature employed in SBWC may cause certain degree of analyte degradation [1–3,10–19]. Therefore, it is important to know whether the solutes are stable under the SBWC conditions employed. Although our previous studies showed degradation of phenanthrene [16], terpene [17] and benzoic acid derivatives [18] in subcritical water under non-chromatographic conditions, it has been reported that some of the pharmaceuticals are stable during high-temperature liquid chromatography runs [15]. Another study revealed that some thiazides experienced degradation during SBWC separation [19]. As mentioned earlier, we have recently developed several SBWC methods for niacinamide, sunscreens and preservatives contained in skincare creams [4–6]. To further evaluate the feasibility of these SBWC methods, we have studied the stability of preservatives in this work. The potential degradation of preservatives was investigated using two different methods. In the first method, peak areas obtained by SBWC at elevated temperatures were compared with those achieved by the conventional HPLC at ambient temperature. Based on previous research [1–3], ZirChrom-DiamondBond-C18, Waters XBridge C18 and Waters XBridge phenyl columns are proved to be more stable at high-temperature conditions. Thus, these three columns were used for SBWC experiments. Tougher conditions were used in the second approach. Mixtures of water–several preservatives were heated for a predetermined period of time, and the amount of preservatives degraded was determined by GC/MS. The temperature involved in both stability studies ranged from 100 to 200°C. To further understand the degradation of preservatives, we identified the degradation products of parabens in water at 200°C. The degradation mechanism was also discussed in this study. Materials and methods Reagents and materials Benzyl alcohol (≥99.0%), methyl paraben (≥99.0%), ethyl paraben (≥99.0%), propyl paraben (≥99.0%) and 2-phenoxyethanol (≥99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Butyl paraben (≥99.0%) was obtained from SAFC (St. Louis, MO, U.S.A.). GD/X PVDF membrane filters (0.45 lm) were received from Whatman (Florham Park, NJ, U.S.A.). HPLC-grade methanol (99.8%) was purchased from Fisher Scientific (Fair Lawn, NJ, U.S.A.). Deionized water (18 MO-cm) was prepared in our laboratory using a Sybron/Barnstead system (Sybron/Barnstead, Boston, MA, U.S.A.). ZirChrom-DiamondBond-C18 (4.6 9 100 mm, 3 lm) column was purchased from ZirChrom Separations, Inc. (Anoka, MN, U.S.A.). XBridge C18 (4.6 9 100 mm, 3.5 lm) and XBridge phenyl (4.6 9 100 mm, 3.5 lm) columns were obtained from Waters Corporation (Milford, MA, U.S.A.). Adsorbosil C18 (4.6 9 150 mm, 5 lm) was acquired from Alltech Associates, Inc. (Deerfield, IL, U.S.A.). Stainless steel vessels (7.07-mL, 9 cm 9 1 cm ID) were purchased from Raleigh Valve and Fitting Company (Raleigh, NC, U.S.A.). Preparation of internal standard solutions For stability studies of the chromatographic evaluation of preservatives, butyl paraben was used as the internal standard. Butyl para-
ben solution was prepared by adding 0.0250 g of butyl paraben to a 50-mL volumetric flask and then diluted to the mark with methanol. For degradation studies on the mixtures of water and multipreservatives, 2-phenoxyethanol was used as the internal standard. However, for studies on mixtures of water and a single paraben, propyl paraben was used as the internal standard. The 2-phenoxyethanol solution was prepared by adding 0.2500 g of 2-phenoxyethanol to a 50-mL volumetric flask and then diluted to the mark with methanol. The propyl paraben solution was prepared by adding 0.1500 g of propyl paraben to a 100-mL volumetric flask and then diluted to the mark with methanol. Preparation of standard solutions For studies on the chromatographic evaluation of preservatives stability under SBWC conditions, a stock standard solution was prepared by adding 0.0750 g of benzyl alcohol and 0.0250 g each of methyl, ethyl and propyl paraben to a 50-mL volumetric flask and then diluted to the mark with methanol. Then, a calibration standard solution was prepared by transferring 2 mL of butyl paraben internal standard solution and 2 mL of the stock standard solution to a 25-mL volumetric flask and then diluted to the mark with methanol. For degradation study of preservatives in the heated water–preservatives mixtures, a stock standard solution was prepared by adding 0.0150 g of benzyl alcohol and 0.0100 g each of methyl, ethyl, propyl and butyl paraben to a 10-mL volumetric flask. Then, 5 mL of water was added to the volumetric flask and then diluted to the mark with methanol. Both this stock solution and the 2-phenoxyethanol internal standard solution were used to prepare three calibration solutions with varying concentrations of preservatives. For degradation study of the water–single paraben mixtures, a stock standard solution was prepared by adding 0.0100 g of p-hydroxybenzoic acid, 0.0100 g of benzoic acid, 0.0150 g of phenol and 0.0100 g of paraben (methyl, ethyl or butyl paraben) to a 10mL volumetric flask. Then, 5 mL of water was added to the volumetric flask and then diluted to the mark with methanol. Both this stock solution and the propyl paraben internal standard solution were used to prepare three calibration solutions with different concentrations of paraben analytes. Heating of water–multipreservatives or water–single paraben mixtures The stainless steel vessels were cleaned with acetone prior to each use. Both ends of each vessel were wrapped with Teflon tape for proper sealing. First, one end of the vessel was tightly sealed with an end cap. For studies on water–multipreservatives mixtures, to each vessel, 0.015 g of benzyl alcohol and 0.010 g each of methyl, ethyl, propyl and butyl paraben were added. For experiments with water–single paraben mixtures, to each vessel, 0.010 g of paraben (methyl or ethyl or butyl paraben) was added. Then, 5 mL of water was added to each vessel. A small amount of void vessel volume was left for thermal expansion of the mixture. Finally, the other end of the loaded vessel was tightly sealed with another end cap. This heating experiment was carried out with four replicates simultaneously. For water–multi preservatives mixtures, the loaded vessels were then heated inside a Fisher Scientific Isotemp Oven (Pittsburg, PA, U.S.A.) at temperatures of 100, 150 or 200°C. At each temperature,
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 306–311
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water–preservatives mixtures were heated for either 30 or 60 min. Similarly, the loaded vessels containing water–single paraben mixtures were heated at 200°C for 30 min. After heating, these vessels were taken out of the oven for cooling. Then, the solution inside each vessel was collected in a 10-mL volumetric flask. To each volumetric flask, 1.00 mL of an appropriate internal standard solution was added and diluted to the mark with methanol. These sample solutions were then filtered through a 0.45-lm Whatman GDX filter into a clean suitable glass vial prior to HPLC analysis on the Adsorbosil C18 column.
Analytical and Consulting Laboratories, Inc. (Detroit, Michigan, U.S.A.) TSSPRO version 3.0 was used for data acquisition and treatment. The GC capillary column used was an Agilent HP-5MS (5% phenyl)-methylpolysiloxane (30 m 9 0.250 mm, 0.25-lm film thickness). The carrier gas was helium and the column flow was 1 mL min1. The injection volume was 1 lL. The injection mode was split and the injector temperature was set at 250°C. The oven temperature profile was as follows: the initial temperature was held at 30°C for 3.00 min. Then, it was increased at 20°C/min to 250°C and held for 5.00 min. The GC interface and the MSD ion chamber were set at 250°C. The MS solvent delay time was 3 min.
SBWC and HPLC analysis Shimadzu Nexera UFLC system (Shimadzu Corporation, Chiyoda-ku Tokyo, Japan) was used for studies on the stability of preservatives. This system has a built-in preheating unit, a column oven and a post-column cooling unit. The column oven can be operated at temperatures up to 160°C. A home-made SBWC system was also employed for stability studies as shown in Fig. 1. A Hitachi L-7100 HPLC pump (Hitachi, Ltd., Tokyo, Japan) is used to deliver the mobile phase. A Valco injector (Valco Instruments Company Inc., Houston, TX, U.S.A.) with a 10-lL loop is connected to the outlet of a preheating coil inside a GC oven (HP 5890 Series 2; Hewlett Packard, Avondale, PA, U.S.A.). The effluent after exiting the oven is cooled with an iced-water bath before entering a Hitachi L-7400 UV detector to protect the UV flow cell. A back pressure regulator (Restek, Bellefonte, PA, U.S.A.) is connected to the outlet of the UV flow cell. The UV detector is connected to a computer via an interface of PC/CHROM (H&A Scientific, Greenville, NC, U.S.A.). Data acquisition and analysis are made available by the PC/CHROM software. For both Shimadzu Nexera UFLC and the home-made system, the wavelength of 256 nm was used for analyte detection. GC/MS analysis GC/MS was used to identify the degradants found in water–single paraben mixtures after heating at 200°C. An Agilent Technologies 6890N Network GC System (Santa Clara, CA, U.S.A.) coupled with a JEOL Ltd. JMS-GC mate II MS System (Tokyo, Japan) was used to confirm the identity of the degradation products. Shrader
Results Chromatographic evaluation of preservatives stability under SBWC conditions To evaluate the stability of preservatives, we compared the average peak areas of the preservatives obtained by SBWC at 150 and 200°C with those achieved by the traditional HPLC at 25°C. The percentage difference in peak areas achieved by SBWC and HPLC is given in Table I. The difference was computed using the formula as described in Table I footnote. Non-chromatographic evaluation of preservatives stability under subcritical water conditions The stability of preservatives was further validated under much tougher conditions by heating the water–multipreservatives mixtures at high temperatures for prolonged period of time. The temperature evaluated ranged from 100 to 200°C, whereas the heating time varied from 30 to 60 min. The percentage recovery of preservatives was calculated using the mass found after heating divided by the mass added before the heating. The results of the non-chromatographic evaluation of preservatives stability are given in Table II. Degradation products of parabens at 200°C The paraben degradation products were investigated by heating the water–single paraben mixtures at 200°C for 30 min. The samples collected after heating were then separated and analysed by GC/
Injector Pump
To Waste Pressure Regulator/ Flow Restrictor
Preheating Coil Column Cooling Unit Water Reservoir
Oven
Detector
Figure 1 Block diagram of a home-made subcritical water chromatography system.
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© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 306–311
B. Kapalavavi et al.
Stability of preservatives in subcritical water Table I Comparison of solute peak areas obtained by subcritical water chromatography (SBWC) at high temperatures and high-performance liquid chromatography (HPLC) at ambient temperature on three columns tested
Butyl paraben
(a) 1 800 000
1 500 000
Percentage difference in peak area (SD)*
Signal
†
‡
XBridge phenyl
XBridge C18
ZirChrom-DB-C18
6 4 8 6
3 4 5 4
12 11 9 11
p-Hydroxy benzoic acid
1 200 000 †
(1) (2) (1) (1)
(1) (2) (4) (3)
(3) (1) (2) (2)
Phenol
900 000
Benzyl alcohol Methyl paraben Ethyl paraben Propyl paraben
600 000
300 000
*Based on five replicates. †
% Difference ¼
4
SBWC Peak Area at 150 C HPLC Peak Area 100 HPLC Peak Area
‡
% Difference ¼
6
8
12
10
14
16
18
Retention time (min)
SBWC Peak Area at 200 C HPLC Peak Area 100 HPLC Peak Area
100
(b)
17.9
80
Relative intensity
Table II Percentage recovery of preservatives found in water–preservatives mixtures after heating at high temperatures Percentage recovery* (SD)†
100 (°C)
150 (°C)
93.6
60
65.5
38.6
40
28.7
200 (°C) 20
Benzyl alcohol Methyl paraben Ethyl paraben Propyl paraben Butyl paraben
30 min
60 min
30 min
60 min
30 min
60 min
99 103 103 103 101
102 103 104 104 101
101 105 104 102 97
101 100 99 97 95
91 89 92 88 88
95 85 89 90 91
(4) (2) (2) (3) (3)
(2) (1) (1) (3) (2)
(1) (1) (1) (1) (2)
(1) (1) (1) (2) (3)
(2) (2) (3) (4) (4)
20
(1) (1) (2) (2) (2)
60
40
80
100
m/z 100
(c)
17.9
80
†
Mass Recovered After Heating %Recovery ¼ 100 Mass Added Before Heating
Relative intensity
*
Based on four replicates.
MS. Figure 2(a) shows the GC chromatogram of a water–butyl paraben mixture after heating at 200°C. The MS spectra of the two unknown degradant peaks are depicted in Fig. 2(b) and (c).
64.6 38.7 92.8
40 137.8
20
Discussion and conclusions As shown in Table I, there is no apparent degradation of analyte for SBWC separations because sometimes SBWC peak areas are slightly larger than that of HPLC and sometimes vice versa. In addition, both t-test and F-test on our results reveal that the peak areas achieved by SBWC and HPLC are not significantly different at the 95% confidence level. It must be pointed out that even if there is a minor degradation of analytes during SBWC runs, the degradation can be compensated by running both calibration standard and sample solutions under the same SBWC conditions. This is because the same degree of analyte degradation should occur for both standard and sample. The results in Table I indicate that SBWC meth-
120.9
60
40
80
120
160
m/z Figure 2 GC/MS chromatogram and mass spectra of a water–butyl paraben mixture heated at 200°C for 30 min. (a) Total ion chromatogram; (b) mass spectrum of the phenol peak; (c) mass spectrum of the p-hydroxybenzoic acid peak.
ods for separation and analysis of preservatives are as reliable as the traditional HPLC methods. The results of the non-chromatographic evaluation of preservatives stability as shown in Table II confirm that the preservatives
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 306–311
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(C6H4(OH)COO)CxHy + H2O
Δ
C6H4(OH)COOH + CxHyOH
Hydrolysis P-hydroxybenzoic acid
Paraben
Δ
H2 O Decarboxylation -CO2
C6H5OH Phenol Figure 3 Paraben degradation pathways.
studied are stable in subcritical water at temperatures up to 150°C. This further validates the conclusion that SBWC methods for preservatives analysis at temperatures up to 150°C are reliable. As shown in Table II, approximately 10% preservatives were degraded at 200°C. Thus, SBWC at 200°C is not recommended for separation and analysis of preservatives. As shown in Fig. 2(a), two sizable degradant peaks were found in the heated water–butyl paraben mixture. These two peaks were identified as phenol and p-hydroxybenzoic acid. The MS spectra of the two peaks are depicted in Fig. 2(b) and (c). Both p-hydroxybenzoic acid and phenol are also confirmed to be the degradants of methyl and ethyl parabens in subcritical water at 200°C. The paraben degradation pathways are illustrated in Fig. 3. The parabens are first degraded into p-hydroxybenzoic acid through hydrolysis reaction and then further into phenol after decarboxylation. Similar paraben degradation mechanism was also reported in the literature by other researchers [20–22].
In closing, degradation of preservatives was only observed in water at 200°C. The major products of paraben degradation in water at 200°C were identified as p-hydroxybenzoic acid and phenol. Based on this result, SBWC methods at 200°C are not recommended for separation and analysis of preservatives. Fortunately, the preservatives studied are stable in subcritical water at temperatures up to 150°C and SBWC within this temperature range is as reliable as the traditional HPLC. Therefore, SBWC at temperatures up to 150°C can be employed for the analysis of preservatives in cosmetic products and other samples. Acknowledgements Part of this work was financially supported by The Procter & Gamble Company. The Shimadzu Nexera UFLC system was acquired through a grant from the Golden LEAF Foundation. The GC/MS was funded by a National Science Foundation MRI grant (0521228). The authors thank H&A Scientific, Inc. for providing the PC/Chrom interface and software.
References 1. Yang, Y. and Kapalavavi, B. Subcritical water chromatography – an economical and green separation technique. Encyclopedia Anal. Chem. doi: 10.1002/9780470027318.a9217, 1–23 (2011). 2. Yang, Y. Subcritical water chromatography: a green approach to high-temperature liquid chromatography. J. Sep. Sci. 30, 1131– 1140 (2007). 3. Smith, R.M. Superheated water chromatography – a green technology for the future. J. Chromatogr. A 1184, 441–455 (2008). 4. Yang, Y., Strickland, Z., Kapalavavi, B., Marple, R. and Gamsky, C. Industrial application of green chromatography – I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase. Talanta 84, 169–174 (2011). 5. Kapalavavi, B., Marple, R., Gamsky, C. and Yang, Y. Separation of sunscreens in skincare creams using greener high-temperature
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drugs by HPLC using superheated water as the mobile phase. Chromatographia 52, S28– S34 (2000). 15. Thompson, J.D. and Carr, P.W. A study of the critical criteria for analyte stability in high-temperature liquid chromato-graphy. Anal. Chem. 74, 1017–1023 (2002). 16. Yang, Y. and Hildebrand, F. Phenanthrene degradation in subcritical water. Anal. Chim. Acta 555, 364–369 (2006). 17. Yang, Y., Kayan, B., Bozer, N., Pate, B., Baker, C. and Gizir, A.M. Terpene degradation and extraction from basil and oregano
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doi: 10.1111/ics.12200
Studies on the stability of preservatives under subcritical water conditions B. Kapalavavi*, R. Marple†, C. Gamsky† and Y. Yang* *Department of Chemistry, East Carolina University, Greenville, NC 27858, and †Global Analytical Capability Organization, The Procter & Gamble Company, Cincinnati, OH 45241, U.S.A.
Received 14 November 2014, Accepted 21 December 2014
Keywords: benzyl alcohol, degradation, high-performance liquid chromatography, parabens, preservatives, skincare creams, stability, subcritical water chromatography
Synopsis OBJECTIVE: The goal of this work was to further validate the subcritical water chromatography (SBWC) methods for separation and analysis of preservatives through the evaluation of analyte stability in subcritical water. METHODS: In this study, the degradation of preservatives was investigated at temperatures of 100–200°C using two different approaches. First, the peak areas obtained by SBWC at high temperatures were compared with those achieved using the traditional high-performance liquid chromatography (HPLC) at 25°C. In the second approach, several preservatives and water were loaded into a vessel and heated at high temperatures for 30 or 60 min. The heated mixtures were then analysed by GC/MS to determine the stability of preservatives. RESULTS: The t- and F-test on the results of the first approach reveal that the peak areas achieved by HPLC and SBWC are not significantly different at the 95% confidence level, meaning that the preservatives studied are stable during the high-temperature SBWC runs. Although the results of the second approach show approximately 10% degradation of preservatives into mainly p-hydroxybenzoic acid and phenol at 200°C, the preservatives studied are stable at 100 and 150°C. This is in good agreement with the validation results obtained by the first approach. CONCLUSION: The findings of this work confirm that SBWC methods at temperatures up to 150°C are reliable for separation and analysis of preservatives in cosmetic and other samples. sume Re OBJECTIF: Le but de ce travail est de valider davantage les methodes de chromatographies d’eau sous-critique (SBWC) pour la separation et l’analyse des conservateurs, par l’evaluation de la stabilite de l’analyte dans l’eau sous-critique. METHODES: Dans cette etude, la degradation des agents de con des temperatures de 100 a 200°C en utiliservation a ete etudiee a sant deux approches differentes. Premierement, les surfaces des pics obtenus par SBWC a des temperatures elevees ont ete comparees 25°C. avec celles obtenues en utilisant la HPLC traditionnelle a Dans la seconde approche, plusieurs agents de conservation et de Correspondence: Yu Yang, Department of Chemistry, East Carolina University, Greenville, NC 27858, U.S.A. Tel.: 1-252-328-9811; fax: 1-252-328-6210; e-mail: [email protected]
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des temperatures l’eau ont ete charges dans une cuve et chauffes a elevees pendant 30 ou 60 min. Les melanges chauffes ont ensuite ete analyses par GC/MS pour determiner la stabilite de conservation. RESULTATS: Les tests t et F sur les resultats de la premiere methode montrent que les surfaces des pics obtenus par HPLC et SBWC ne sont pas significativement differentes au seuil de confiance de 95%, ce qui signifie que les agents conservateurs etudies sont stables pendant l’execution de la SBWC a haute temperature. Bien que les resultats de la seconde approche montrent environ 10% de degradation des conservateurs vers principalement de l’acide p-hydroxybenzo€ıque et de phenol a 200°C, les conservateurs etudies sont stables entre 100 et 150°C. Ceci est en bon accord avec les resultats de la validation obtenus par la premiere methode. CONCLUSION: Les resultats de ces travaux confirment que les des temperamethodes de chromatographie de l’eau subcritique a tures allant jusqu’ a 150°C sont fiables pour la separation et l’analyse des conservateurs dans les cosmetiques et d’autres echantillons. Introduction Preservatives are used in over 22 000 cosmetic products with concentration of up to 0.4% for a single paraben. Obviously, preservative concentrations in cosmetic products must be accurately determined before the products can be released. The most common technique for the analysis of preservatives is high-performance liquid chromatography (HPLC). However, HPLC requires a huge amount of organic solvents for its mobile phase. These organic solvents used in HPLC mobile phase are not only hazardous and expensive in purchasing, but also require waste disposal that further increases the costs. Fortunately, pure water at elevated temperatures can be used as the mobile phase to achieve reversedphase separations [1–9]. We have recently developed subcritical water chromatography (SBWC) methods for separation and analysis of niacinamide, sunscreens and preservatives in skincare products [4–6]. Our work demonstrated that SBWC can achieve the same analytical quality as that obtained by HPLC, thus eliminating the hazardous and expensive organic solvents required in HPLC. The temperature required in SBWC separations depends on the polarity of analytes as well as the property of the stationary phase. Please note that SBWC is a technique of reversed-phase liquid chromatography where the mobile phase is more polar than the stationary phase. In general, less polar solutes require less polar
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
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mobile phase to be efficiently eluted. This in turn requires higher temperature that decreases the polarity of subcritical water used as the mobile phase in SBWC. However, the high temperature employed in SBWC may cause certain degree of analyte degradation [1–3,10–19]. Therefore, it is important to know whether the solutes are stable under the SBWC conditions employed. Although our previous studies showed degradation of phenanthrene [16], terpene [17] and benzoic acid derivatives [18] in subcritical water under non-chromatographic conditions, it has been reported that some of the pharmaceuticals are stable during high-temperature liquid chromatography runs [15]. Another study revealed that some thiazides experienced degradation during SBWC separation [19]. As mentioned earlier, we have recently developed several SBWC methods for niacinamide, sunscreens and preservatives contained in skincare creams [4–6]. To further evaluate the feasibility of these SBWC methods, we have studied the stability of preservatives in this work. The potential degradation of preservatives was investigated using two different methods. In the first method, peak areas obtained by SBWC at elevated temperatures were compared with those achieved by the conventional HPLC at ambient temperature. Based on previous research [1–3], ZirChrom-DiamondBond-C18, Waters XBridge C18 and Waters XBridge phenyl columns are proved to be more stable at high-temperature conditions. Thus, these three columns were used for SBWC experiments. Tougher conditions were used in the second approach. Mixtures of water–several preservatives were heated for a predetermined period of time, and the amount of preservatives degraded was determined by GC/MS. The temperature involved in both stability studies ranged from 100 to 200°C. To further understand the degradation of preservatives, we identified the degradation products of parabens in water at 200°C. The degradation mechanism was also discussed in this study. Materials and methods Reagents and materials Benzyl alcohol (≥99.0%), methyl paraben (≥99.0%), ethyl paraben (≥99.0%), propyl paraben (≥99.0%) and 2-phenoxyethanol (≥99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Butyl paraben (≥99.0%) was obtained from SAFC (St. Louis, MO, U.S.A.). GD/X PVDF membrane filters (0.45 lm) were received from Whatman (Florham Park, NJ, U.S.A.). HPLC-grade methanol (99.8%) was purchased from Fisher Scientific (Fair Lawn, NJ, U.S.A.). Deionized water (18 MO-cm) was prepared in our laboratory using a Sybron/Barnstead system (Sybron/Barnstead, Boston, MA, U.S.A.). ZirChrom-DiamondBond-C18 (4.6 9 100 mm, 3 lm) column was purchased from ZirChrom Separations, Inc. (Anoka, MN, U.S.A.). XBridge C18 (4.6 9 100 mm, 3.5 lm) and XBridge phenyl (4.6 9 100 mm, 3.5 lm) columns were obtained from Waters Corporation (Milford, MA, U.S.A.). Adsorbosil C18 (4.6 9 150 mm, 5 lm) was acquired from Alltech Associates, Inc. (Deerfield, IL, U.S.A.). Stainless steel vessels (7.07-mL, 9 cm 9 1 cm ID) were purchased from Raleigh Valve and Fitting Company (Raleigh, NC, U.S.A.). Preparation of internal standard solutions For stability studies of the chromatographic evaluation of preservatives, butyl paraben was used as the internal standard. Butyl para-
ben solution was prepared by adding 0.0250 g of butyl paraben to a 50-mL volumetric flask and then diluted to the mark with methanol. For degradation studies on the mixtures of water and multipreservatives, 2-phenoxyethanol was used as the internal standard. However, for studies on mixtures of water and a single paraben, propyl paraben was used as the internal standard. The 2-phenoxyethanol solution was prepared by adding 0.2500 g of 2-phenoxyethanol to a 50-mL volumetric flask and then diluted to the mark with methanol. The propyl paraben solution was prepared by adding 0.1500 g of propyl paraben to a 100-mL volumetric flask and then diluted to the mark with methanol. Preparation of standard solutions For studies on the chromatographic evaluation of preservatives stability under SBWC conditions, a stock standard solution was prepared by adding 0.0750 g of benzyl alcohol and 0.0250 g each of methyl, ethyl and propyl paraben to a 50-mL volumetric flask and then diluted to the mark with methanol. Then, a calibration standard solution was prepared by transferring 2 mL of butyl paraben internal standard solution and 2 mL of the stock standard solution to a 25-mL volumetric flask and then diluted to the mark with methanol. For degradation study of preservatives in the heated water–preservatives mixtures, a stock standard solution was prepared by adding 0.0150 g of benzyl alcohol and 0.0100 g each of methyl, ethyl, propyl and butyl paraben to a 10-mL volumetric flask. Then, 5 mL of water was added to the volumetric flask and then diluted to the mark with methanol. Both this stock solution and the 2-phenoxyethanol internal standard solution were used to prepare three calibration solutions with varying concentrations of preservatives. For degradation study of the water–single paraben mixtures, a stock standard solution was prepared by adding 0.0100 g of p-hydroxybenzoic acid, 0.0100 g of benzoic acid, 0.0150 g of phenol and 0.0100 g of paraben (methyl, ethyl or butyl paraben) to a 10mL volumetric flask. Then, 5 mL of water was added to the volumetric flask and then diluted to the mark with methanol. Both this stock solution and the propyl paraben internal standard solution were used to prepare three calibration solutions with different concentrations of paraben analytes. Heating of water–multipreservatives or water–single paraben mixtures The stainless steel vessels were cleaned with acetone prior to each use. Both ends of each vessel were wrapped with Teflon tape for proper sealing. First, one end of the vessel was tightly sealed with an end cap. For studies on water–multipreservatives mixtures, to each vessel, 0.015 g of benzyl alcohol and 0.010 g each of methyl, ethyl, propyl and butyl paraben were added. For experiments with water–single paraben mixtures, to each vessel, 0.010 g of paraben (methyl or ethyl or butyl paraben) was added. Then, 5 mL of water was added to each vessel. A small amount of void vessel volume was left for thermal expansion of the mixture. Finally, the other end of the loaded vessel was tightly sealed with another end cap. This heating experiment was carried out with four replicates simultaneously. For water–multi preservatives mixtures, the loaded vessels were then heated inside a Fisher Scientific Isotemp Oven (Pittsburg, PA, U.S.A.) at temperatures of 100, 150 or 200°C. At each temperature,
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 306–311
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water–preservatives mixtures were heated for either 30 or 60 min. Similarly, the loaded vessels containing water–single paraben mixtures were heated at 200°C for 30 min. After heating, these vessels were taken out of the oven for cooling. Then, the solution inside each vessel was collected in a 10-mL volumetric flask. To each volumetric flask, 1.00 mL of an appropriate internal standard solution was added and diluted to the mark with methanol. These sample solutions were then filtered through a 0.45-lm Whatman GDX filter into a clean suitable glass vial prior to HPLC analysis on the Adsorbosil C18 column.
Analytical and Consulting Laboratories, Inc. (Detroit, Michigan, U.S.A.) TSSPRO version 3.0 was used for data acquisition and treatment. The GC capillary column used was an Agilent HP-5MS (5% phenyl)-methylpolysiloxane (30 m 9 0.250 mm, 0.25-lm film thickness). The carrier gas was helium and the column flow was 1 mL min1. The injection volume was 1 lL. The injection mode was split and the injector temperature was set at 250°C. The oven temperature profile was as follows: the initial temperature was held at 30°C for 3.00 min. Then, it was increased at 20°C/min to 250°C and held for 5.00 min. The GC interface and the MSD ion chamber were set at 250°C. The MS solvent delay time was 3 min.
SBWC and HPLC analysis Shimadzu Nexera UFLC system (Shimadzu Corporation, Chiyoda-ku Tokyo, Japan) was used for studies on the stability of preservatives. This system has a built-in preheating unit, a column oven and a post-column cooling unit. The column oven can be operated at temperatures up to 160°C. A home-made SBWC system was also employed for stability studies as shown in Fig. 1. A Hitachi L-7100 HPLC pump (Hitachi, Ltd., Tokyo, Japan) is used to deliver the mobile phase. A Valco injector (Valco Instruments Company Inc., Houston, TX, U.S.A.) with a 10-lL loop is connected to the outlet of a preheating coil inside a GC oven (HP 5890 Series 2; Hewlett Packard, Avondale, PA, U.S.A.). The effluent after exiting the oven is cooled with an iced-water bath before entering a Hitachi L-7400 UV detector to protect the UV flow cell. A back pressure regulator (Restek, Bellefonte, PA, U.S.A.) is connected to the outlet of the UV flow cell. The UV detector is connected to a computer via an interface of PC/CHROM (H&A Scientific, Greenville, NC, U.S.A.). Data acquisition and analysis are made available by the PC/CHROM software. For both Shimadzu Nexera UFLC and the home-made system, the wavelength of 256 nm was used for analyte detection. GC/MS analysis GC/MS was used to identify the degradants found in water–single paraben mixtures after heating at 200°C. An Agilent Technologies 6890N Network GC System (Santa Clara, CA, U.S.A.) coupled with a JEOL Ltd. JMS-GC mate II MS System (Tokyo, Japan) was used to confirm the identity of the degradation products. Shrader
Results Chromatographic evaluation of preservatives stability under SBWC conditions To evaluate the stability of preservatives, we compared the average peak areas of the preservatives obtained by SBWC at 150 and 200°C with those achieved by the traditional HPLC at 25°C. The percentage difference in peak areas achieved by SBWC and HPLC is given in Table I. The difference was computed using the formula as described in Table I footnote. Non-chromatographic evaluation of preservatives stability under subcritical water conditions The stability of preservatives was further validated under much tougher conditions by heating the water–multipreservatives mixtures at high temperatures for prolonged period of time. The temperature evaluated ranged from 100 to 200°C, whereas the heating time varied from 30 to 60 min. The percentage recovery of preservatives was calculated using the mass found after heating divided by the mass added before the heating. The results of the non-chromatographic evaluation of preservatives stability are given in Table II. Degradation products of parabens at 200°C The paraben degradation products were investigated by heating the water–single paraben mixtures at 200°C for 30 min. The samples collected after heating were then separated and analysed by GC/
Injector Pump
To Waste Pressure Regulator/ Flow Restrictor
Preheating Coil Column Cooling Unit Water Reservoir
Oven
Detector
Figure 1 Block diagram of a home-made subcritical water chromatography system.
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Stability of preservatives in subcritical water Table I Comparison of solute peak areas obtained by subcritical water chromatography (SBWC) at high temperatures and high-performance liquid chromatography (HPLC) at ambient temperature on three columns tested
Butyl paraben
(a) 1 800 000
1 500 000
Percentage difference in peak area (SD)*
Signal
†
‡
XBridge phenyl
XBridge C18
ZirChrom-DB-C18
6 4 8 6
3 4 5 4
12 11 9 11
p-Hydroxy benzoic acid
1 200 000 †
(1) (2) (1) (1)
(1) (2) (4) (3)
(3) (1) (2) (2)
Phenol
900 000
Benzyl alcohol Methyl paraben Ethyl paraben Propyl paraben
600 000
300 000
*Based on five replicates. †
% Difference ¼
4
SBWC Peak Area at 150 C HPLC Peak Area 100 HPLC Peak Area
‡
% Difference ¼
6
8
12
10
14
16
18
Retention time (min)
SBWC Peak Area at 200 C HPLC Peak Area 100 HPLC Peak Area
100
(b)
17.9
80
Relative intensity
Table II Percentage recovery of preservatives found in water–preservatives mixtures after heating at high temperatures Percentage recovery* (SD)†
100 (°C)
150 (°C)
93.6
60
65.5
38.6
40
28.7
200 (°C) 20
Benzyl alcohol Methyl paraben Ethyl paraben Propyl paraben Butyl paraben
30 min
60 min
30 min
60 min
30 min
60 min
99 103 103 103 101
102 103 104 104 101
101 105 104 102 97
101 100 99 97 95
91 89 92 88 88
95 85 89 90 91
(4) (2) (2) (3) (3)
(2) (1) (1) (3) (2)
(1) (1) (1) (1) (2)
(1) (1) (1) (2) (3)
(2) (2) (3) (4) (4)
20
(1) (1) (2) (2) (2)
60
40
80
100
m/z 100
(c)
17.9
80
†
Mass Recovered After Heating %Recovery ¼ 100 Mass Added Before Heating
Relative intensity
*
Based on four replicates.
MS. Figure 2(a) shows the GC chromatogram of a water–butyl paraben mixture after heating at 200°C. The MS spectra of the two unknown degradant peaks are depicted in Fig. 2(b) and (c).
64.6 38.7 92.8
40 137.8
20
Discussion and conclusions As shown in Table I, there is no apparent degradation of analyte for SBWC separations because sometimes SBWC peak areas are slightly larger than that of HPLC and sometimes vice versa. In addition, both t-test and F-test on our results reveal that the peak areas achieved by SBWC and HPLC are not significantly different at the 95% confidence level. It must be pointed out that even if there is a minor degradation of analytes during SBWC runs, the degradation can be compensated by running both calibration standard and sample solutions under the same SBWC conditions. This is because the same degree of analyte degradation should occur for both standard and sample. The results in Table I indicate that SBWC meth-
120.9
60
40
80
120
160
m/z Figure 2 GC/MS chromatogram and mass spectra of a water–butyl paraben mixture heated at 200°C for 30 min. (a) Total ion chromatogram; (b) mass spectrum of the phenol peak; (c) mass spectrum of the p-hydroxybenzoic acid peak.
ods for separation and analysis of preservatives are as reliable as the traditional HPLC methods. The results of the non-chromatographic evaluation of preservatives stability as shown in Table II confirm that the preservatives
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 37, 306–311
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(C6H4(OH)COO)CxHy + H2O
Δ
C6H4(OH)COOH + CxHyOH
Hydrolysis P-hydroxybenzoic acid
Paraben
Δ
H2 O Decarboxylation -CO2
C6H5OH Phenol Figure 3 Paraben degradation pathways.
studied are stable in subcritical water at temperatures up to 150°C. This further validates the conclusion that SBWC methods for preservatives analysis at temperatures up to 150°C are reliable. As shown in Table II, approximately 10% preservatives were degraded at 200°C. Thus, SBWC at 200°C is not recommended for separation and analysis of preservatives. As shown in Fig. 2(a), two sizable degradant peaks were found in the heated water–butyl paraben mixture. These two peaks were identified as phenol and p-hydroxybenzoic acid. The MS spectra of the two peaks are depicted in Fig. 2(b) and (c). Both p-hydroxybenzoic acid and phenol are also confirmed to be the degradants of methyl and ethyl parabens in subcritical water at 200°C. The paraben degradation pathways are illustrated in Fig. 3. The parabens are first degraded into p-hydroxybenzoic acid through hydrolysis reaction and then further into phenol after decarboxylation. Similar paraben degradation mechanism was also reported in the literature by other researchers [20–22].
In closing, degradation of preservatives was only observed in water at 200°C. The major products of paraben degradation in water at 200°C were identified as p-hydroxybenzoic acid and phenol. Based on this result, SBWC methods at 200°C are not recommended for separation and analysis of preservatives. Fortunately, the preservatives studied are stable in subcritical water at temperatures up to 150°C and SBWC within this temperature range is as reliable as the traditional HPLC. Therefore, SBWC at temperatures up to 150°C can be employed for the analysis of preservatives in cosmetic products and other samples. Acknowledgements Part of this work was financially supported by The Procter & Gamble Company. The Shimadzu Nexera UFLC system was acquired through a grant from the Golden LEAF Foundation. The GC/MS was funded by a National Science Foundation MRI grant (0521228). The authors thank H&A Scientific, Inc. for providing the PC/Chrom interface and software.
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