j. Soc. Cosmet. Chem., 31,323-327 (November 1980) Analysis of N-nitrosodiethanolamine in linoleamide DEA by high pressure liquid chromatography and UV detection IRA E. ROSENBERG, JOHN GROSS, and TONY SPEARS, Clairol Incorporated, 2 Blachley Road, Stamford, Ct. 06902 Received May 14, 1980. Synopsis A method for the analysis of N-nitrosodiethanolamine in Linoleamide DEA which uses a simple sample preparation and MICRO-SAMPLING TECHNIQUES is presented. The nitrosamine is first separated from the bulk of the matrix by normal phase high pressure liquid chromatography followed by quantitation using reverse phase HPLC. Absolute limit of detection is ! ng. INTRODUCTION For the past several years, the Cosmetic, Toiletry and Fragrance Association (CTFA) has been sponsoring contract research for the development of nitrosamine analytical methodology. The main area of concentration has been the development of methodol- ogy for the analysis of N-nitrosodiethanolamine (NDE1A) in cosmetic raw materials with the use of high pressure liquid chromatography and detection using a Thermal Energy Analyzer (TEA). Methodology for the determination of NDE1A by HPLC-UV detection was also developed and validated. Although most of the alkanolamides of diethanolamine lent themselves to HPLC-TEA methodology, Linoleamide DEA was an exception. The major problem in the development of methodology was a large TEA responding peak appearing just before the NDE1A elution area when normal phase HPLC was used. Reverse phase HPLC using TEA detection was not attempted, because good separations on reverse phase packings are usually obtained with highly aqueous solvent systems. The TEA is not compatible with highly aqueous solvent systems, thus eliminating reverse phase HPLC separation. Rosenberg and co-workers (1), as well as Rahn and Mitchell (2), have reported on the analysis of NDE1A using reverse phase HPLC with UV detection. Likewise, Rosen- berg and co-workers (3) have recently developed a two column HPLC-UV method for the micro-isolation and quantitation of NDE1A by HPLC-UV in various cosmetic raw materials and finished product. In this note, we wish to present the applicability of this method for the determination of NDE1A in Linoleamide DEA. 323

324 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS EXPERIMENTAL APPARATUS A Waters Associates (Milford, MA) Model 6000A solvent delivery system equipped with a Model U6K injector and Model 440 absorbance detector fixed at 254 nm is used throughout the method. A Spectra Physics (Piscataway, NJ) Model SP4100 computing integrator is used for quantitation. The columns are a Waters Assoc. /.tPorasil column (4 mm I.D. x 30 cm) and a Whatman Inc. (Clifton, NJ) Partisil PXS 10/25 ODS (C•8) (4.6 mm I.D. x 25 cm). Pierce Chemical Co. (Rockford, IL) "Reactivials" are used for collection vessels. SOLVENTS The liquid chromatographic solvents were obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ) and are "HPLC Reagent" grade methanol and chloroform. The distilled water was obtained from the Electrified Water Co. (Newark, NJ). All solvents are filtered using a Millipore filtration apparatus. The N-nitrosodiethanolamine (NDE1A) was obtained from Columbia Chemical Co. (Columbia, SC). CHROMATOGRAPHIC CONDITIONS Table I illustrates the conditions for each of two columns utilized. PREPARATION OF STANDARD AND SAMPLE A NDE1A standard was prepared by weighing 50 mg of NDE1A into a 100-ml flask and bringing to volume with a solution of CHC13/MeOH (95/5) (Standard A). A working standard for HPLC was prepared by pipetting ! ml of Standard A into a 100-ml flask and bringing to volume with CHCI3/MeOH (95/5) (Standard B). This working standard has a concentration of 5 ppm or 5 ng//al. Linoleamide DEA samples were prepared by weighing 2.5-3.0 grams into a 10-ml flask and bringing to volume with CHCI3/MeOH (95/5). PROCEDURE Inject 5/.tl of Standard B into/.tPorasil column to establish elution volume of NDE1A and collect NDE1A area. Inject 50/.d of sample and collect NDE1A area. Evaporate Table I Chromatographic Conditions for Silica and C•8 Columns Columns /xPorasil (silica) Partisil PXS(C•8 ) Mobile Phase 95/5 (chloroform/methanol) V/V H20 (100%) Flow Rate 1 ml/min 1 ml/min Chart Speed 10 mm/min 10 mm/min Detector Sensitivity 0.1-2.0 aufs 0.005 aufs