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).

NITROSAMINE ANALYSIS 243 6. To determine total amount of NDE1A present in the original cocamide DEA sample, use the following equation: ppm (in sample) = ppm (found) x 100/.t.__l x 10 _ml (25/.tl) (sample wt. in grams) ' E. DETERMINATION OF NDE1A IN COCAMIDE DEA USING AN RCSS HPLC Parameters for initial separation of NDEIA.' Column--Radial Pak B, Waters Associates, 7.9 cm I.D. x 10 cm (normal phase packing). Mobile Phase--30:70 Tetrahydrofuran/methylene chloride. Flow Rate--7 ml/min. Pressure (operating range)--O-2500 psi. Detector sensitivity--O.02 aufs. Recorder a) Range--10 my. b) Chart speed--10 ram/min. HPLC Parameters for Quantitation of NDEIA.' Column--Radial Pak B, Waters Associates, 7.9 cm I.D. x 10 cm (normal phase packing). Mobile Phase--water. Flow Rate--4 ml/min. Pressure (operating range)--O-2500 psi. Detector sensitivity--O.005 aufs. Recorder a) Range--10 rev. b) Chart speed--10 ram/min. Procedure 1. Weigh 30 g of cocamide DEA into a 50-ml volumetric flask and dilute to volume with methanol/water (1:1). Shake well until cocamide DEA dissolves. 2. Pipet 5 ml of the sample into a 10-ml volumetric flask. Dilute to volume with methanol/water (1:2) and shake thoroughly. This gives a sample that is 30% W/V cocamide DEA dissolved in approximately (40:60) methanol/water. 3. Inject 50 3tl of the sample and the 1 ng/3tl standard NDE1A solution into the LC which contains a Radial Pak B cartridge equilibrated with 30:70 tetrahydrofuran/ methylene chloride mobile phase. 4. Collect the elution volume for the NDE1A peak in the standard and sample. A 5-ml collection volume assures that no sample is lost. 5. Evaporate the collected samples to dryness using nitrogen and reconstitute the samples and standard in 400/.tl of water. 6. Inject 25/zl of the standard and the sample solutions into the LC using a Radial Pak A cartridge equilibrated with water. Record the response for the NDElA peak for both the sample and standard.