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.

244 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 7. To determine the quantity of NDEIA present in the cocamide DEA, use the following equations: ng NDE1A standard injected ng NDEIA = Response of NDEIA standard x Response of NDE1A in Sample, ppb NDEIA ng found x 50 ml x 10 ml x 400 •tl x 1000 wt. of sample (g) x 5 ml x 50/al x 25/al F. ANALYSIS OF FINISHED PRODUCT USING RCSS HPLC Parameters for the initial separation and quantitation of NDEIA can be found under Section E of this experimental section. Procedure The preparation of NDEIA standards and the preparation of a standard linearity curve for NDE1A can be found under "Method of analysis for cocamide DEA," Section D in this experimental section, except that the diluting solvent and mobile phase for the Radial Pak B is 30:70 tetrahydrofuran/methylene chloride. The mobile phase for the Radial Pak A cartridge is water. Sample Preparation.' 1. Weigh 2-3 g of sample into a 10-ml volumetric flask. 2. Add 3 ml of the THF (UV grade) and swirl until the sample dissolves. Dilute to volume with the same solvent. Determination of NDEIA in Finished Products.' 1. Make 25-/xl injection of standard on a Radial Pak B column to determine retention volume of NDE1A. 2. Make 25-/xl injection of sample and collect the area corresponding to the retention volume of NDE1A. 3. Evaporate each collection to dryness and reconstitute in 100/xl of 95:5 water/metha- nol. 4. Make 25-/xl injection of the reconstituted collected sample onto a Radial Pak A column using water as the eluting solvent, 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 NDEIA data points). 6. To determine total amount of NDE1A present in the original sample, use the