Anal. Chem. 2007, 79, 2096-2100
Quantitative 13C NMR Spectroscopic Studies on the Equilibrium of Formaldehyde with Its Releasing Cosmetic Preservatives Detlef Emeis,*,† Willem Anker,‡ and Klaus-Peter Wittern†
Beiersdorf AG, Unnastrasse 48, D-20245 Hamburg, Germany, and Bode-Chemie, Melanchthonstrasse 27, D-22525 Hamburg, Germany
The aim of our study was to show that NMR spectroscopy is an excellent method to obtain reliable information about the equilibrium between free formaldehyde and its formaldehyde releasers. For this purpose, we compared several O- and N-formal-based formaldehyde releasers used in industrial and consumer products. The underlying chemical structures as well as the release of formaldehyde were followed quantitatively as a function of the pH and dilution. It was shown that only the amide-based Nformals are a reservoir for formaldehyde in the concentrations normally used in cosmetic products, whereas O-formals and the amine-based N-formals decompose completely. Since NMR spectroscopy does not affect the equilibrium between free and bound formaldehyde, we think that it is the only method for unequivocal determination of free formaldehyde. Measurements on finished products showed that free formaldehyde can be quantified down to concentrations as low as ∼0.002 wt % in an acceptable measuring time. Formaldehyde releasers continue to find use as components of chemical disinfectants and as preservatives in industrial and consumer products. Exposure to formaldehyde is, however, subject to restrictions because of its toxicological properties. Annex VI of Cosmetic Directive 76/768/EC stipulates, All finished products containing formaldehyde or substances in this Annex and which release formaldehyde must be labeled with the warning “contains formaldehyde” where the concentration of formaldehyde in the finished product exceeds 0.05 wt %.1 Reliable methods for quantification of free formaldehyde are therefore of great interest. The group of formaldehyde releasers can be divided into O-formals and N-formals. N-formals can be amine- or amide-based. In O-formals, formaldehyde and the starting substance are linked via oxygen and in the N-formals via nitrogen:
O-formal: N-formal:
formaldehyde
starting substance
releaser
HCHO HCHO
+ ROH h + R1R2NH h
RO-CH2-OH R1R2N-CH2-OH
The underlying addition reactions are reversible. Therefore, some free hydrated formaldehyde (methylene glycol) will always * To whom correspondence should be addressed. E-mail: Detlef.Emeis@ Beiersdorf.com. Fax: +49 40 4909 183913. † Beiersdorf AG. ‡ Bode-Chemie.
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be present in aqueous solution. At higher concentrations, admixtures of oligomers of formaldehyde and of hemiformals with different chain lengths must be taken into account. If the formaldehyde is consumed, the formaldehyde releaser supplies new formaldehyde. The action of formaldehyde-releasing agents is therefore based on the continual attainment of this equilibrium. Studies in our laboratory revealed2-4 that structural information on commercially available cosmetic formulations having many ingredients can also be obtained by means of NMR spectroscopy. It is essential that this method can measure sample volumes in the cubic centimeter range without influencing the system studied and especially any chemical equilibria that may be present. Since cosmetic products usually contain large amounts of water, particularly 13C NMR spectroscopy is suitable for determination of the structure and concentration of the ingredients. The aim of the studies described here was to compare by means of NMR spectroscopy several O- and N-formals used in the cosmetic industry. Of interest were the underlying chemical structures as well as the release of formaldehyde as a function of pH and dilution. Many different methods are available for the qualitative and quantitative analysis of formaldehyde. The best known method is probably one based on the reaction of formaldehyde with acetylacetone and ammonia with subsequent colorimetric determination of the diacetyldihydrolutidine formed.5 Along with methods based on titrimetric, photometric, or chromatographic procedures, NMR investigations on the pure formaldehyde/water system are also known. Le Botlan et al.6 presented the 1H- and 13C-chemical shifts of the various oligomers, thereby renewing the work of de Breet et al.,7 and studied the temperature- and concentration-dependent equilibria between monomeric and oligomeric formaldehyde in aqueous solution. Dilution leads to depolymerization which, at 0 °C, can take up to 50 h. At (1) Eighth Commission Directive 86/199/EEC of 26 March 1986 adapting to technical progress Annexes II, IV and VI to Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. Official J. L 149, 03/06/1986; pp 0038-0045. (2) Plass, J.; Emeis, D.; Blu ¨ mich, B. J. Surf. Deterg. 2001, 4, 379-384. (3) Berg, T.; Arlt, P.; Brummer, R.; Emeis, D.; Kulicke, W.-M.; Wiesner, S.; Wittern, K.-P. Colloids Surf., A 2004, 238, 59-69. (4) Alberola, C.; Blu ¨ mich, B.; Emeis, D.; Wittern, K.-P. Colloids Surf., A 2006, 290, 247-255. (5) Nash, T. Biochem. J. 1965, 5, 416-421. (6) Le Botlan, D. J.; Mechin, B. G.; Martin, G. J. Anal. Chem. 1983, 55, 587591. (7) de Breet, A. J. J.; Dankelman, W.; Huysmans, W. G. B.; de Wit, J. Angew. Makromol. Chem. 1977, 62, 7-31. 10.1021/ac0619985 CCC: $37.00
© 2007 American Chemical Society Published on Web 01/24/2007
formaldehyde concentrations below ∼0.5 wt %, monomeric formaldehyde is the only observable species. Albert et al.8 observed the formation of the oligomers via 13C NMR spectra within minutes after transferring gaseous formaldehyde into water circulating through a 13C flow-through cell. Maiwald et al.9 and Ott et al.10 developed a thermodynamic model of the equilibria between the different oligomers, relying on quantitative online 1H NMR in a flow-through cell as a function of time after dilution. At room temperature and neutral pH, the time course of the attainment lies in the range of minutes. Quantitative determination of the free formaldehyde simultaneously with that bound in a formaldehyde releaser is a clearly greater challenge. The chromatographic separation developed by Engelhardt and Klinkner11 with subsequent derivatization based on the acetylacetone reaction named above was the precursor of the official analytical procedure of the EU for cosmetic products, which was modified in 1990.12 Despite many advances in analysis and the development of other measuring methods,13 quantitative determination of free formaldehyde simultaneously with bound formaldehyde at low concentrations in solutions, but especially in finished products, remains a problem with no satisfactory answer.14 A clear disadvantage of these analytical methods5,11-14 is the fact that sooner or later they influence the formaldehyde releaser h formaldehyde equilibrium; although temperature or pH fluctuations as well as reaction and column contact times can be minimized, they cannot be neglected. We show in this work that 13C NMR spectroscopy offers an excellent solution to this problem because, as a purely physical method with a high spectral resolution and sufficient sensitivity, it does not, as already mentioned, affect the equilibrium between formaldehyde and the formaldehyde releaser during the measurement. As we are interested in equilibrium conditions especially at low concentrations of formaldehyde which, in turn, lead to long measuring times, we did no systematic time-dependent studies. Orienting experiments show that the attainment can take several hours. MATERIALS AND METHODS Sodium hydroxymethylglycinate and diazolidinyl urea were obtained from International Specialty Products (ISP) (Wayne, NJ), imidazolidinyl urea was from 3V Sigma (Bergamo, Italy), DMDM hydantoin was from Lonza (Basel, Switzerland), and benzylhemiformal was from Lanxess (Leverkusen, Germany). The finished product Eucerin Q 10 Active Night Cream came from routine production of Beiersdorf AG (Hamburg, Germany). The measurements were performed on a Bruker DPX 250 NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) in a 10-mm multinuclear probe at room temperature with no locking. (8) Albert, K.; Peters, B.; Bayer, E. Z. Naturforsch., B 1986, 41, 351-358. (9) Maiwald, M.; Fischer, H. H.; Kim, Y.-K.; Albert, K.; Hasse, H. J. Magn. Reson. 2004, 166, 135-146. (10) Ott, M.; Fischer, H. H.; Maiwald, M.; Albert, K.; Hasse, H. Chem. Eng. Process. 2005, 44, 653-660. (11) Engelhardt, H.; Klinkner, R. Chromatographia 1985, 20, 559-565. (12) Commission Directive 90/207/EEC of 4 April 1990 amending the Second Directive 82/434/EEC on the approximation of the laws of the Member State relating to methods of analysis necessary for checking the composition of cosmetic products. Official J. L 108, 28/04/1990; pp 0092-0101. (13) Rivero, R. T.; Topiwala, V. J. Cosmet. Sci. 2004, 55, 343-350. (14) Karlberg, A.-T.; Skare, L.; Lindberg, I.; Nyhammar, E. Contact Dermatitis 1998, 38, 20-28.
The solvents for the formaldehyde releasers were aqueous citrate or borate buffer solutions from Merck (Darmstadt, Germany). Preparation and measurement were in general separated by a time lag of several hours. For a rough estimation of the attainment of the equilibrium in selected samples, the NMR measurements were started immediately after preparation. To shorten the measuring time for 13C NMR spectroscopy, especially at low concentrations of the releasers and pH values lower than ∼8, the gadolinium salt of diethylenetriaminepentaacetic acid was added as a relaxation reagent. Because this reagent shifts the pH itself, no addition was possible at pH values above ∼8. The finished products were measured as neat samples without any additions. Between 100 and 40 000 proton decoupled free induction decays with 13C pulse widths of 90° and spectral widths of 240 ppm were added. With relaxation reagent added, the repetition times were 5 s or otherwise 10-20 s. In selected samples, the intensities of the (protonated) carbon signals of interest increase only slightly with increasing repetition times between 10 and 20 s. So we think that within a relative measuring error of 25%, our quantifications are reliable. If necessary, the multiplicities of the individual carbon signals were determined using DEPT135 and DEPT90 sequences. The hydrated formaldehyde showed the characteristic signal of a CH2 group at 82 ppm (tetramethylsilane ) 0 ppm). The concentrations of free formaldehyde and its releasers were in some cases determined relative to the previously measured concentrations of the citric acid of the buffer using the integrals of the corresponding signals and in others relative to a defined amount of spiked acetone. In finished products they were determined relative to the known concentrations of the ingredients in the formulas. For determination of the concentrations of different reaction products of the formaldehyde releasers, the sum of the identified molecules was set equal to 100%. RESULTS AND DISCUSSION Serving as an example for the O-formals was benzylhemiformal, the reaction product of formaldehyde and benzyl alcohol, although it is no longer widely used in cosmetic products. Sodium hydroxymethylglycinate was studied as the representative of the amine-based N-formals. Examples for the amide-based N-formals were DMDM hydantoin, which is by far the most widely used formaldehyde releaser in the cosmetic industry, as well as diazolidinyl urea and imidazolidinyl urea. Benzylhemiformal. The neat sample contained 95 wt % active substance and 5 wt % water. Quantitative analysis of the NMR spectra revealed that the benzylhemiformal consisted of 40 mol % benzyl alcohol and 60 mol % formaldehyde, with the reaction products consisting of 1 mol of benzyl alcohol and several moles of formaldehyde present in addition to the 1:1 molar product. In the undiluted sample, ∼20 mol % of the substance was free benzyl alcohol. Unbound formaldehyde formed mainly oligomers. Due to the low water solubility of benzylhemiformal, no homogeneous mixtures of this product can be prepared with concentrations higher than ∼5 wt %. A mixture of 5 wt % benzylhemiformal and 95 wt % water can be prepared in 1 h by shaking. About 90% of the benzylhemiformal in this mixture by then has decomposed. Figure 1 shows the 13C spectrum for this concentration. The signals of the aromatic CH groups can be seen at 128 ppm as well as two different quaternary C signals between Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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Figure 1. 13C spectrum of benzylhemiformal in a concentration of 5 wt % in water. Signals 1, 1′, 2, 2′, and 3 as given in the structures; arrow, free hydrated formaldehyde; signal at 86 ppm, formaldehyde dimer.
135 and 140 ppm originating from benzyl alcohol (2′) and from benzylhemiformal with one methylol group (2). The alcohol group of the free benzyl alcohol is seen at 64 ppm (1′) and that of the hemiformal at 69 ppm (1); at 88 ppm, the methylol group of the hemiformal can be seen (3). The free hydrated formaldehyde shows its signal at 82 ppm (arrow); the signal at 86 ppm arises from the formaldehyde dimer remaining as the only oligomer in comparison with the neat sample because of dilution.6 Mixing of the appropriate amounts of water, formaldehyde, and benzyl alcohol at room temperature leads within ∼1 h exactly to this composition of a 5 wt % aqueous solution of benzylhemiformal. This clearly shows that free formaldehyde forms an equilibrium with the releaser benzylhemiformal. A 2 wt % solution of benzylhemiformal in water mixes spontaneously, with decomposition of 97% of the benzylhemiformal. Higher dilution leads to instantaneous, complete decomposition of the releaser into formaldehyde and benzyl alcohol. At the maximum permissible concentration of 0.15 wt % benzylhemiformal in cosmetic rinse-off products, the concentration of free formaldehyde is consequently just under 0.05 wt %. Because this releaser is of such little importance for cosmetics, a possible pH dependence of the above results was not studied. Sodium Hydroxymethylglycinate. The first N-formal-based product studied was sodium hydroxymethylglycinate. As mentioned previously, it is an amine-based formaldehyde releaser. For the commercially available 50 wt % solution, we determined a free formaldehyde concentration of less than 0.002 wt % using NMR spectroscopy. At a concentration of ∼1 wt % and a pH of 5.5, sodium hydroxymethylglycinate decomposes completely into formaldehyde and sodium glycinate; in a basic milieu, decomposition is incomplete. It is not until further dilution to ∼0.5 wt %sthe maximum concentration allowed in cosmetic productss that it decomposes completely at a pH of 8.5. The concentration of free formaldehyde is then 0.12 wt %. The corresponding equilibria are attained quickly. Figure 2 shows a typical 13C spectrum for a pH of 8.5-9 and a concentration of 1 wt %. The signals of the citric acid buffer system appear at 182, 179, 75, and 46 ppm (*). Free hydrated formaldehyde can again be seen at 82 ppm (arrow). The starting substance sodium hydroxymethylglycinate has a signal for the 2098 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
Figure 2. 13C spectrum of sodium hydroxymethylglycinate in a concentration of 1 wt % in water, buffered with citric acid (signals with asterisks) to pH 8.5-9. Signals 1, 1′, 2, 2′, and 3 as given in the structures; arrow, free hydrated formaldehyde.
Figure 3. Structural formulas of 1,3-dimethylol-5,5-dimethylhydantoin and reaction products resulting from release of formaldehyde. The signals belonging to the numbered atoms are shown in Figure 4.
carbonyl function at 177 ppm (1), for the NCH2COO group at 56 ppm (2), and for the methylol group at 72 ppm (3). The signals for sodium glycinate appear at 173 (1′) and 42 ppm (2′). Forty mole percent of the starting substance had decomposed, resulting in a free formaldehyde concentration of 0.09 wt %. 1,3-Dimethylol-5,5-dimethylhydantoin. One mole of the amide-based formaldehyde releaser 1,3-dimethylol-5,5-dimethylhydantoin (DMDM hydantoin) can release a total of 2 mol of formaldehyde. Figure 3 presents the possible structures that can be found in different ratios in aqueous solution depending on the dilution and pH. Figure 4 shows a typical 13C spectrum of DMDM hydantoin 25 wt % in water at pH 6. In the full spectrum, the two chemically nonequivalent carbonyl signal groups can be seen at 180 and 156 ppm, the methyl groups at 23 ppm, free hydrated formaldehyde at 82 ppm (arrow), and the signals of the quaternary carbon atoms and the methylol groups at 62 ppm. This range is shown expanded in the inset. The methylol signals of A are at 63.0 (2) and 61.6 ppm (3) and the quaternary carbon of A is at 62.1 ppm (1). The methylol signal of B is at 61.3 ppm (3′) and the quaternary carbon at 59.3 ppm (1′). C has its methylol signal at 62.7 ppm (2′′) and its quaternary carbon at 63.4 ppm (1′′). The concentration of D is below the limit of detection at this dilution. Table 1 shows the molar distribution of the four components, the molar content of bound and free formaldehyde, and the
Table 2. Diazolidinyl Urea and Imidazolidinyl Urea: Concentrations of Free Formaldehyde Compared with the Maximum Releasable Concentrations of Formaldehyde as a Function of Dilution and pH
active ingredient (wt %) FA free (wt %): pH 9 pH 6 pH 4 max rel FA (wt %)
Figure 4. 13C spectrum of 1,3-dimethylol-5,5-dimethylhydantoin in a concentration of 25 wt % in water, pH 6. Inset: expansion of the spectrum between 59 and 64 ppm. Signals 1, 1′, 1′′, 2, 2′′, 3, and 3′ as given in the structures in Figure 3; arrow, free hydrated formaldehyde. Table 1. Concentrations of DMDM Hydantoin and Its Reaction Products (Figure 3) as Well as of Bound and Free Formaldehyde (FA) as a Function of Dilution and pH pH 8.5-9: active ingredient (wt %) 25 5 structure A (mol %) 86 77 structure B (mol %) 10 18 structure C (mol %) 4 5 structure D (mol %) nd nd FA bound (mol %) 93 81 FA free (mol %) 7 19 FA free (wt %) 0.53 0.32
0.5 28 32 22 18 54 46 0.073
0.25 0.08 0.05 17 nda nd 33 35 33 24 17 14 26 48 53 44 18 23 56 82 77 0.047 0.027 0.013
5.1 71 19 7 3 87 13 0.21
0.6 50 22 23 5 66 34 0.071
pH 6-6.5: active ingredient (wt %) 55 25 structure A (mol %) 88 86 structure B (mol %) 8 9 structure C (mol %) 4 5 structure D (mol %) nd nd FA bound (mol %) 98 96 FA free (mol %) 2 4 FA free (wt %) 0.39 0.33
0.25 39 12 39 10 63 37 0.027
0.11 30 8 52 10 59 41 0.014
pH 4-4.5: active ingredient (wt %) 25 4.9 0.5 0.25 0.1 structure A (mol %) 88 80 57 44 27 structure B (mol %) 8 8 6 10 9 structure C (mol %) 4 12 37 46 64 structure D (mol %) nd nd nd nd nd FA bound (mol %) 96 91 78 69 61 FA free (mol %) 4 9 22 31 39 FA free (wt %) 0.28 0.14 0.036 0.021 0.013 and,
not detectable.
concentration of free formaldehyde as a function of pH and dilution. In alkaline solution, equilibrium is attained quickly; in acidic solution, it is attained mainly at low concentrations below ∼1 wt % over a period of several hours. It can be seen from Table 1 that, at the pH normally found in cosmetic products and concentrations in the 0.2 wt % range, this N-formal-based formaldehyde releaser, unlike sodium hydroxymethylglycinate and the O-formal-based benzyl hemiformal, is always a reservoirsalbeit smallsfor bound formaldehyde. Addition of free formaldehyde to 5 wt % DMDM hydantoin at pH 6 increases the concentration of free formaldehyde but not
diazolidinyl urea 0.5 0.2 0.084 0.036 0.032 0.21
0.047 0.019 0.024 0.086
imidazolidinyl urea 0.6 0.2 0.024 0.012 0.015 0.14
0.015 0.011 0.010 0.046
by an amount corresponding to the amount of formaldehyde added. Instead, the concentration of component A increases as well, accompanied by a change in the composition of A-D. Addition of 5,5-dimethylhydantoin (structure D) to 5 wt % DMDM hydantoin at pH 6 likewise does not result in the expected calculated increase in the concentration of D; instead, the composition changes, with a reduction in the concentration of free formaldehyde observed. This is further proof of the presence of equilibria between free formaldehyde and these types of formaldehyde releasers. Diazolidinyl Urea and Imidazolidinyl Urea. These formaldehyde releasers are complex mixtures of several condensation products of formaldehyde and allantoin for which full structural elucidation is very difficult.15 Therefore, for these formaldehyde releasers, we determined only the free formaldehyde concentration at different pH for the approximate concentrations used and for the maximum permissible concentrations of these substances in cosmetic products. Table 2 presents the results. The manufacturer produces diazolidinyl urea from 4 mol of formaldehyde and 1 mol of allantoin, imidazolidinyl urea from 3 mol of formaldehyde and 2 mol of allantoin.16 This leads to the maximum amounts of releasable formaldehyde given in Table 2. It is evident that a reservoir of bound formaldehyde is always present at the chosen pH values and dilutions. Finished Cosmetic Product. We routinely measure the concentration of free formaldehyde in our own products by NMR spectroscopy. Figure 5 shows a typical 13C spectrum of a neat sample of Eucerin Q 10 Active Night Cream that has been preserved with 0.15 wt % DMDM hydantoin. The CH2 signal of the hydrated formaldehyde at 82 ppm can be clearly seen in the inset. Its intensity corresponds to a free formaldehyde concentration of ∼0.013 wt %. The signals of the different components of the formaldehyde releaser DMDM hydantoin (see structures A-D above) cannot usually be identified in the spectra due to the many signals and the resulting overlapping of signals. The pH of the emulsion was between 5.5 and 6.5. In view of the fact that the finished product is a complex mixture of the widest variety of substances having an unknown influence on the equilibrium between formaldehyde and the formaldehyde releaser, the measured concentration of free formaldehyde is in line with (15) Lehmann, S. V.; Hoeck, U.; Breinholdt, J.; Olsen, C. E.; Kreilgaard, B. Contact Dermatitis 2006, 54, 50-58. (16) Merianos, J. J.; Sondossi, M.; Wachocki, B. A.; Rossmoore, H. W. In Conference Proceedings Preservatech; 27-28 May 1998; pp 29-38 (Verlag fu ¨r chemische Industrie H. Ziolkowsky GmbH.).
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Figure 5. 13C spectrum of a neat sample of Eucerin Q 10 Active Night Cream from day-to-day production. Inset: expansion of the spectrum between 79 and 85 ppm.
the results for the pure substance DMDM hydantoin (Table 1). The limit of detection for an acceptable measuring time of ∼15 h on a 250-MHz NMR instrument is ∼0.002 wt % in finished products; this can be reduced by increasing the measuring time and/or field intensity. SUMMARY AND CONCLUSIONS Our studies confirm that 13C NMR spectroscopy can be used as a noninvasive method for determination of the free formaldehyde concentration. Especially in finished products, the limit of detection is low enough to be able to measure formaldehyde
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concentrations much lower than the legally required duty of declaration of 0.05 wt %. The method does not interfere with the chemical equilibria and is therefore suitable for studies on formaldehyde releasers in water. Such studies show that the free formaldehyde concentration particularly in amide-based N-formals in the concentrations used in cosmetic formulations is clearly below the theoretical total content of chemically bound formaldehyde. Therefore, these formaldehyde releasers form reservoirs for formaldehyde. Oformals and amine-based N-formals already release all of their bound formaldehyde at moderate dilutions corresponding to concentrations clearly higher than those used in cosmetic products. Therefore, they may just as well be replaced with pure formaldehyde. Studies on the pH dependence showed that sodium hydroxymethylglycinate tends to release formaldehyde more in acidic solutions and DMDM hydantoin, diazolidinyl urea, and imidazolidinyl urea more in alkaline solutions. Attainment of equilibrium is usually rapid but can take up to several hours. Addition of free formaldehyde or formaldehyde-free starting material at room temperature changes the relative ratios of the components in DMDM hydantoin. Likewise at room temperature, benzylhemiformal forms spontaneously when benzyl alcohol and formaldehyde are mixed in water. Thus, the reactions of the starting substances and formaldehyde are equilibrium reactions.
Received for review December 22, 2006. AC0619985
October
24,
2006.
Accepted
