NITROSAMINE ANALYSIS 241 e) Hold time--15-20 min. f) Reverse time (final condition to initial condition)--10 min. g) Final condition--60% Solvent A/40% Solvent B (allow approximately 30 min. for equilibration between runs). h) Programming is not needed for NDEIA standard runs, use initial condition (a). The preparation of NDEIA standards and the preparation of a standard linearity curve for NDEIA can be found under "Method of Analysis for Ethanolamines" in this experimental section, except that the diluting sovent is 60/40, water/methanol (V/V). Sample Preparation.' 1. Weigh 2.0-2.5 gm of sample into a 10-ml volumetric flask. 2. Add 3 ml of 60/40 water/methanol (V/V) and swirl until sample dissolves, then dilute to mark with 60/40 water/methanol. Determination of NDEIA in lauramide DEA.' 1. Inject 25/zl of 60/40 water/methanol to check for any interferences. 2. Inject 25/zl of sample solution. Program the instrument after NDE1A peak is eluted (refer to Solvent Program). 3. Record peak height (cm) of NDE1A in sample. 4. To determine the concentration (ppm) of NDEIA present in the sample, refer to Part 4 under "Determination of NDE1A in di- and triethanolamines" found in this experimental section. D. METHOD OF ANALYSIS FOR COCAMIDE DEA (USING A TWO COLUMN SEPARATION) HPLC parameters for initial separation of NDEIA.' Column/zPorasil, Waters Associates, 3.9 mm I.D. x 30.0 cm, stainless steel. Mobile phase--95%/5% chloroform/methanol V/V. Flow rate--l.O ml/min. Pressure (operating range)--O-6000 psi. Detector sensitivity--O.02 aufs. Recorder a) Range--10 mv. b) Chart speed--10 ram/min. HPLC parameters for NCEL4 quantitation on !aBondapak ( C•8).' Column/zBondapak (C•8). Flow rate--1 ml/min. Mobile phase--95%/5% water/methanol (V/V). Pressure (operating range)--O-6000 psi. Detector sensitivity--O.005 aufs. Recorder a) Range--10 mv. b) Chart speed--10 ram/min.

242 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Procedure Preparation of N-nitrosodiethanolamine (NDElA ) standards.' 1. Weigh (0.08-0.1 g) of NDEIA into 100-ml volumetric flask. 2. Add 30 ml of 95/5 chloroform/methanol V/V, swirl until dissolved and dilute to mark with the same solvent mixture. 3. Pipet 1 ml of stock solution into 100-ml volumetric flask and dilute to mark to give working stock solution of 10 ppm (8-12 ppm) NDEIA. 4. Prepare the following standards: 1.2, 1.0, 0.8 ppm by pipeting appropriate mls of working stock solution into volumetric flasks and diluting to volume with 95/5 chloroform/methanol. Preparation of standard linearity curve r or NDElA: 1. Inject 25 •tl of stock solution (10 ppm) to determine retention volume of NDE1A. 2. Inject 25 •tl of standards in duplicate in a •tPorasil column and elute with 95/5 chloroform/methanol. 3. Collect the NDE1A peak and evaporate each collection to dryness, and reconstitute in 100/al of 95/5 water/methanol (V/V). 4. Inject (in duplicate) 25/al of the reconstituted sample into a/aBondapak C•8 column and elute with 95/5 water/methanol. 5. Record the peak height (cm) of each standard. 6. Take the average peak height of the standards and plot the peak height (cm) vs. the concentration (ppm). A linear curve should be obtained. 7. Calculate slope and y-intercept of the linearity curve by a regression line analysis of the total standard data points. Sample preparation.' 1. Weigh 2.0-3.0 gm into a 10-ml volumetric flask. 2. Add 3 ml of 95/5% chloroform/methanol (V/V) and swirl until dissolved. Dilute to mark with the same solvent. Determination of NDEIA in cocamide DEA.' 1. Make 25 •tl injection of standard on a •tPorasil column to determine retention volume of NDEIA. 2. Make 25 •tl injection of sample and collect the area corresponding to the retention volume of NDEIA. 3. Evaporate each collection to dryness and reconstitute in 100 •tl of 95/5 water/ methanol. 4. Make 25 •tl injection of reconstituted collected sample and reinject onto a •tBondapak C•8 column, and record peak height (cm) of NDEIA in sample. 5. To determine the concentration (ppm) of NDEIA present in the reconstituted collected sample, use the following equation: y =ax+b y = concentration of NDE1A present in the reconstituted collected sample a = slope x = peak height in (cm) b = y-intercept (a and b were calculated from standard NDE1A data points).