J. Soc. Cosmet. Chem., 42, 97-104 (March/April 1991) Gas chromatographic determination of 1,4-dioxane at the parts-per-million level in consumer shampoo products MARK P. ITALIA and MATHEWS A. NUNES, Johnson & Johnson Consumer Products Company, Grandview Road, Skillman, NJ 08558. Received November 16, 199. Synopsis A packed-column gas chromatographic method for the determination of 1,4-dioxane in shampoo products is presented. The sample shampoo is diluted with water containing isobutanol as the internal standard and injected directly into a gas chromatograph containing a 12-foot X /,-in stainlessteel column packed with OV-1 as the stationary phase. The reproducibility of the method is better than 7% relative standard deviation, with 1,4-dioxane recoveries between 94 and 105%, and is linear over the range of 1 ppm to 250 ppm. The entire analysis can be performed in 15 minutes, allowing for fast sample turnaround. The ruggedness of the method has been demonstrated by the analysis of 13 shampoos, 12 containing ethoxylated surfactants and 1 containing no ethoxylates. All 12 products containing ethoxylated surfactants tested positive for 1,4-dioxane, with the range of 1,4-dioxane found being from 6 ppm to 144 ppm. INTRODUCTION Within the shampoo manufacturing sector of the cosmetics industry there is concern about the level of residual 1,4-dioxane present in final shampoo products. The 1,4- dioxane is introduced into the product via the use of ethoxylated fatty alcohol sulfates as cleansing agents. During the process of alcohol ethoxylation, ethylene oxide can dimerize to form 1,4-dioxane, which is subsequently carried through the shampoo manufacturing process. This compound has been shown to be carcinogenic animals (1,2) and is listed as a carcinogen with NIOSH (3). Concern over the health risks, along with the probability that the ethoxylated surfactants in use contain a fair amount of residual 1,4-dioxane, has lead many cosmetic and toiletry companies to examine the levels of dioxane in both their raw materials and their finished products. Although many producers have begun vacuum stripping procedures in their manufacture of the fatty alcohol sulfates, there can still be high levels of 1,4-dioxane remaining in the materials that will be inadvertently added into finished product. Mark P. Italia's current address is Knoll Pharmaceuticals, Inc., 30 North Jefferson Road, Whippany, NJ 07981. 97
98 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Currently, 1,4-dioxane is most commonly analyzed by gas chromatographic methods (4-7). However, these methods require extensive sample pretreatment and have high variability and relatively poor recoveries. An HPLC method has been developed by Scalia (8), yet from the quality control aspects of time and complexity, this method suffers from the relative difficulty of using a gradient HPLC method. Additionally, Rastogi (9) has developed a headspace gas chromatographic-mass spectroscopic analysis method using mass spectral selectivity to identify and quantitate dioxane. While this method is acceptable in a research laboratory environment, it suffers in that mass spectroscopic capabilities do not exist in most quality control laboratories. This paper describes a gas chromatographic method for the analysis of 1,4-dioxane in shampoos. The method is rapid, requires no sample pretreatment, requires minimal sample preparation, and utilizes an internal standard for quantitation. EXPERIMENTAL REAGENTS AND MATERIALS HPLC grade 1,4-dioxane was purchased from Aldrich Chemical Company (Milwaukee, WI), and reagent grade (ACS certified) isobutanol was obtained from Fisher Scientific (Fairlawn, NJ). Double deionized water was distilled and deionized (Millipore, Inc., Medford, MA). Sampling vials were from Wheaton Laboratory Products (Millville, N J) and were sealed with crimp tops and teflon-lined septa. INTERNAL STANDARD DILUENT An isobutanol internal standard diluent was prepared based upon expected values of 1,4-dioxane in the shampoo products. A 2000-ppm stock solution of isobutanol in water was prepared in a crimp-top vial. This solution was further diluted to produce a final working diluent solution of 20 ppm. CALIBRATION STANDARD SOLUTION A standard calibration solution was prepared and analyzed to determine the relative response factor of the isobutanol internal standard to the 1,4-dioxane. A 1,4-dioxane stock solution was prepared at a level of 2000 ppm, and an aliquot was used to prepare an approximate 20 ppm solution. 1.0 ml of this solution was then added to 1.0 ml of the isobutanol diluent solution, resulting in a calibration standard of 10 ppm 1,4- dioxane and 10 ppm isobutanol. This calibration standard was then analyzed using the gas chromatographic program described below. SAMPLE SOLUTIONS Approximately 1 to 2 grams of shampoo sample were weighed into a sampling vial, and an equal weight of isobutanol diluent was added. The vial was crimp-sealed and shaken gently until complete dispersion of the sample was achieved. If foaming occurred, the
DETERMINATION OF 1,4-DIOXANE 99 Table I Reproducibility of Response Ratio Area isobutanol Area 1,4-dioxane Response ratio 147436 56600 2.60 148348 59739 2.48 156260 60161 2.60 159059 60291 2.64 164563 65101 2.53 163548 63199 2.59 159638 63356 2.52 154759 62834 2.46 Average response ratio = 2.55. Standard deviation = 0.06. Relative standard deviation = 2.5%. sample was allowed to sit until it dissipated (approximately 15 minutes). This sample solution was analyze directly using the gas chromatographic program described below. GAS CHROMATOGRAPHIC CONDITIONS The gas chromatographic system was a Hewlett Packard 5890A equipped with a flame 1.50e+6-5.00e. y = - 1.B7e+4 + 5046.gx R^2 = o.ggg8 0.00 +0 50 100 150 200 250 i PPM DIOXANE Figure 1. Linearity of 1,4-dioxane standards from 1 ppm to 250 ppm.
100 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ionization detector. The output signal was sent to a Nelson Analytical data system. The column used was a 12-foot x Vs-in stainless steel packed column of 15% OV-1 on 100/120 Chromasorb WHP (Supelco, Inc., Bellefonte, PA). The injector temperature was 185øC and the detector temperature was 325øC. The column temperature program was: 85øC for 2 minutes, followed by a linear temperature increase of 5øC/min to 95øC, with a hold of 1 minute. This was followed by a cleanup step involving a temperature increase of 25øC/min to a temperature of 300øC, where it was held for 5 minutes. The helium carrier flow was 40 ml/min, and a 1-1 direct injection was made into a quartz injection port liner loosely packed with silanized glass wool. RESULTS AND DISCUSSION It was necessary to determine if a sample prepared by this method would be homogeneous and if it could be reproducibly analyzed. To confirm this, a sample of Shampoo B was prepared in the prescribed manner and analyzed nine times to determine the reproducibility of the response ratio. The resulting response ratios were determined as the area of isobutanol compared to the area of 1,4-dioxane, and are tabulated in Table I. The response ratio for this test mixture was determined to be 2.55-0.06 with a relative standard deviation of 2.5%. This confirms that the sample preparation results in a homogeneous mixture. y= 13.8+ 0.9x R^2 =0.9996-20 - 1 0 ß I ' I ' I ' I 0 10 20 30 40 PPM DIOXANE SPIKED Figure 2. Standard addition of 1,4-dioxane into Shampoo B.
DETERMINATION OF 1,4-DIOXANE 101 Studies were performed to determine the linear range and recovery of the method. 1,4-dioxane standards were prepared at levels between 1 and 250 ppm and analyzed. The results are graphically presented as FID response vs ppm 1,4-dioxane in Figure 1. The area/concentration curve showed a correlation coefficient of 0.9998, indicating linearity over the concentration range examined. Since we were unable to obtain a 1,4-dioxane-free shampoo that contained ethoxylated surfactants, standard addition experiments were performed on two different shampoos to determine the linearity and recovery of the system. One of the shampoos (Shampoo B) contained ethoxylated compounds, while a second shampoo (Shampoo K) contained no ethoxylated surfactants. A spiking solution of 250 ppm 1,4-dioxane in water was prepared and different volumes were spiked into samples of both shampoos. These samples were then prepared and analyzed according to the procedure outlined previously. The resultant data is graphically displayed in Figures 2 and 3. Shampoo B had a linear response over the spiking range of 0 to 40 ppm, with a correlation coefficient of 0.9996, and providing an intercept of 13.8 ppm 1,4-dioxane. This result compares favorably with an independent analysis of the same sample, which determined the level of 1,4-dioxane in Shampoo B to be 13.0 ppm. As shown in Figure 3, analysis of Shampoo K, the ethoxylate-free shampoo, was linear over the spiking concentration range of 7 to 225 ppm 1,4-dioxane, with a correlation coefficient of 0.9999 and an intercept of 1.5 ppm. This also compares favorably with an independent analysis that determined the level of 1,4-dioxane in Shampoo K to be 1.3 ppm. The recovery of the 20O 100 ß. 0.9999 o -50 I ' I '! 50 150 250 PPM DIOXANE SPIKED Figure 3. Standard addition of 1,4-dioxane into Shampoo C.
102 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Recovery of 1,4-Dioxane Spiked Into Shampoo K and Shampoo B 1,4-Dioxane 1,4-Dioxane added (ppm) found (ppm) % Recovery Shampoo B Shampoo K 0 13.0 N.A. 9.3 22.0 98 14.4 27.2 98 21.2 33.4 94 38.9 51.1 94 0 1.5 N.A. 5.7 7.5 104 18.1 19.3 98 3O.2 33.6 105 91.9 98.1 105 164.0 173.6 105 216.3 229.6 105 spiked 1,4-dioxane from both shampoos is shown in Table II. The results indicate that the recovery of 1,4-dioxane from these shampoos is between 94 and 105%. This variability is most likely due to the difficulty in accurately analyzing compounds at such trace levels. SAMPLE ANALYSIS Thirteen shampoos were purchased on the open market and analyzed for 1,4-dioxane content by this method. All but one of the shampoos indicated at least one ethoxylated compound on its list of ingredients. One shampoo (Shampoo K) was chosen due to the lack of any ethoxylates among its ingredients, and was used as a blank. The results of the analysis of these shampoos are presented in Table III. As can be seen, there is great variation in the level of 1,4-dioxane present in shampoos containing ethoxylated surfactants. The lowest level of 1,4-dioxane found was 6 ppm, while the highest 1,4- Table III Levels of 1,4-Dioxane Found in 13 Shampoos Shampoo 1,4-Dioxane (ppm) A 6 B 13 C N.A. D 144 E 53 F 16 G 112 H 7 I 67 J 39 K 1 L 42 M 47 N.A.: Not analyze due to chromatographic interferences.
DETERMINATION OF 1,4-DIOXANE 103 dioxane level was 144 ppm. These differences are most likely due to the amount of ethoxylated components in the shampoo as well as to varying amounts of 1,4-dioxane in the ethoxylated surfactants. Figure 4 presents a typical chromatogram of a shampoo containing a low level of 1,4-dioxane. Replicates of different preparations of the same shampoo agreed within 7% of each other (in the worst case). Analyses performed on a ISOBUTANOL 1,4-DIOXANE. I 1 2,q. TIME (MINUTES) Figure 4. Gas chromatogram of Shampoo A. GC conditions: Initial temperature: 85øC; initial time: 2 minutes; temperature gradient A: 5øC; final temperature A: 95øC; final hold:! minute; temperature gradient B: 25øC; final temperature B: 300øC; injector temperature: 250øC; detector temperature: 325øC; helium carrier flow: 40 ml/minute; injection size: 1 ul; column: 12' X ¾8" stainless steel column packed with 15% OV-1 on Supelco WHP.
104 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS second column of differing lot number also provided the same results, including the 7% variability in 1,4-dioxane level. In 12 of the 13 shampoos the analyseshowed that no interferences were present that would impact upon the determination of the 1,4-dioxane level. However, in the case of Shampoo C, there was an unknown interference that occurred. This interference partially co-eluted with the isobutanol internal standard and provided for a variability of greater than 50%, thus eliminating the shampoo from the study. CONCLUSION An internal standard method utilizing isobutanol as the internal standard has been shown to be capable of accurately analyzing for 1,4-dioxane in a variety of commercial shampoos. The method is linear and provides recoveries between 94 and 105% over a 1,4-dioxane concentration range of 1 to 250 ppm. Thirteen shampoos were analyzed for 1,4-dioxane content. The lowest level of 1,4-dioxane was found in a shampoo containing no ethoxylated materials. The lowest concentration of 1,4-dioxane in an ethoxylatecontaining shampoo was 6 ppm and the highest was 144 ppm. REFERENCES (1) C. Hoch-Legeti, M. F. Argus, and J. C. Arcos, Introduction of carcinomas the nasal cavity of rats by dioxane, Br. J. Cancer, 24, 164-167 (1970). (2) R.J. Kociba, S. B. McCollister, C. Park, T. R. Torkelson, and P. J. Gehring, 1,4-dioxane. I. 2-Year ingestion study in rats, Toxicol. Appl. Pharmacol., 30, 275-286 (1974). (3) NIOSH, Registry of Toxic œfj$cts of Chemical Substances, 1986 edition, NIOSH Publication No. 87-114, 3, 2082-2083 (1987). (4) D. B. Black, R. C. Lawrence, E. G. Lovering, and J. R. Watson, Gas-liquid chromatographic method for determining 1,4-dioxane in cosmetics, J. Assoc. Off. Anal. Chem., 66, 181-183 (1983). (5) T. J. Birkel, C. R. Warner, and T. Fazio, Gas chromatographic determination of 1,4-dioxane in Polysorbate 60 and Polysorbate 80, J. Assoc. Off. Anal. Chem., 62, 931-936 (1979). (6) J. J. Robinson and E. W. Ciurczak, Direct gas chromatographic determination of 1,4-dioxane in ethoxylated surfactants,j. Soc. Cosmet. Chem., 31, 329-337 (1980). (7) B. A. Waldman, Analysis of 1,4-dioxane in ethoxylated compounds by gas chromatography/mass spectrometry using selected ion monitoring, J. Soc. Cosmet. Chem., 33, 19-25 (1982). (8) S. Scalia, M. Guarneri, and E. Menegatti, Determination of 1,4-dioxane in cosmetic products by high performance liquid chromatography, Analyst, 115, 929-931 (1990). (9) S. C. Rastogi, Headspace analysis of 1,4-dioxane in products containing polyethoxylated surfactants by GC-MS, Chromatographia, 29, 441-445 (1990).