j. Soc. Cosmet. Chem., 31,219-222 (July/August 1980) Nitrosamine determination by use of conventional equipment VICTOR H. BAPTIST, Ph.D. and RICHARD BROWN, B.S., Max Factor & Co. 1655 N. Mc Cadden Place, Hollywood, CA 90028. Received January 11, 1980. Presented at the Annual Scientific Meeting, Society of Cosmetic Chemists, December 6-7, 1979, New York, New York. Synopsis The determination of NITROSAMINES in consumer products has heretofore been hampered by the need for costly specialized equipment. An effective technique to analyze for total nonvolatile nitrosamines has been developed which utilizes only conventional equipment. The method is as specific as the thermal energy analyzer. We are able to achieve a sensitivity of better than one part per billion. Six to seven analyses can be completed each day by one technician. In addition, the technique is also adaptable to determining if NITRITES are present in raw materials. We have found the method successful in use on cosmetic products, grilled hot dogs, beer and cutting fluids. INTRODUCTION Nitrosamines have attracted an increasing amount of attention of cosmetic manufac- turers due to the disclosure by Fine in 1977 of their presence in cosmetics. The thermal energy analyzer has been the method of specific nitrosamine detection due to its ability of detecting nitrosamines at parts per billion. Industry needed an equally sensitive method that was less costly. The colorimetric technique that follows which detects total nitrosamines met these goals. One technician can complete 6 to 7 samples per day. In addition the technique is also adaptable to determine "nitrite" present in raw materials. EXPERIMENTAL APPARATUS AND REAGENTS All work was performed utilizing a Beckman DGB or Hitachi 100-60 spectrophotom- eter, vacuum oven, vacuum pump, ultrasonic cleanser, 4 oz. glass bottles with teflon liners, and 8 ml glass vials plus standard chemical glassware. Standard N-nitrosodiethanolamine (NDE1A) was obtained from Columbia Organic Chemical Co. (Columbia, South Carolina) 1-naphthylamine-7-sulfonic acid from Pfaltz 219

220 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and Bauer (Stamford, Conn.) Hi-Flosil 60/200 mesh from Applied Science Lab Inc. anhydrous methanol, acetone and ethylene dichloride-all three distilled in glass from Burdick and Jackson sulfanilic acid, anhydrous sodium sulfate power, glacial acetic acid (aidehyde-free), hydrobromic acid, 48% and bromine, all A.R. grade from Baker or Mallinckrodt. All chemicals were used as received. Solutions used for analysis were prepared as follows: standard NDEIA solution--0.1% NDEIA in anhydrous methanol solution S is 0.75 gm sulfanilic acid plus 50 ml glacial acetic acid, q.s. to 250 ml with distilled water solution N is 0.3 gm 1-naphthylamine- 7-sulfonic acid plus 50 ml glacial acetic acid, q.s. to 250 ml with distilled water color solution is an equal volume of solutions S & N hydrobromic acid bromine solution use 62.5 ml of 48% hydrobromic acid plus 2 ml bromine, q.s. with glacial acetic acid to i liter. Store all solutions in dark or brown bottles and cap tightly since the nitrogen oxides in the air develop the color. Do not use color solution unless it is colorless. The glassware is all initially treated with 6 N hydrochloric acid to remove adsorbed nitrogen oxide from its surface and then put thru the entire assay process before actually being used for assays. Bottles and vials remained sealed until the day they are to be used. Before use, bottles and vials are cleaned by rinsing at least three times each with tap water, then distilled water and finally with acetone and then dried in the vacuum oven. The column is prepared by using a wad of glass wool, 10 gm Hi Flosil is added and gently tamped to level the surface, 25 gm anhydrous sodium sulfate is added. The assembled column is washed with 200 ml of acetone before use. METHOD OF ANALYSIS The assay procedure utilizes the well known Griess Test as modified by Ilosvay for nitrite using Cleve's acid (1-naphthylamine-7-sulfonic acid) (1) which is similar to the color procedure used in the nitrite method authored by Rosenberg, et al. (2). By increasing sample size one is able to achieve a sensitivity of better than one part per billion. Thus with a sample size of 2.5 gm a sensitivity of 3-5 ppb is attained. Further sensitivity is proportional to increased sample size. A 5-gm sample plus 2.5 ml of water is extracted and concentrated--using 100 ml of acetone for extraction. Extraction is performed by placing the sample flask into an ultrasonic bath for 30 minutes. Sulfanilic acid (0.25 gin) is added to the acetone-water during extraction to destroy any nitrite present. This extract is dried and cleaned up by passing it thru an acetone prewashed anhydrous sodium sulfate-silica gel column. The 125 ml of acetone extract which is collected is divided in half. One sample of each pair is "spiked" with 5 ug NDEIA to: 1) determine recovery, 2) be certain nothing is present in the extract that prevents color development. One milliliter of glacial acetic acid is added to each portion of the acetone extract to insure destruction of any nitrite that may not have been earlier destroyed (3,4,5). Each portion is evaporated to dryness by means of a steam bath in a 4 oz. screw cap bottle. The extract is taken to dryness in a vacuum oven heated at 90øC with at least 125-250 torr (20-25 inches of vacuum). Since additional cleanup is needed, the residue is dissolved in one milliliter of water and this solution is extracted with 10 ml of ethylene dichloride. The ethylene dichloride is removed by means of a Pasteur pipette. The aqueous extract is taken to dryness in the vacuum oven. To the residue in the 4 oz.