NITROSAMINE ANALYSIS 247 which elutes very close to NDE1A. When analyzing these samples, extreme care should be taken to wash and recondition the column between runs, as described in the Experimental Section, to eliminate any interferences resulting from this component. Figure 1 illustrates the chromatographic separation achieved for triethanolamine spiked with approximately 100 and 150 ppb NDEIA. ANALYSIS OF ALKANOLAMIDES Alkanolamides manufactured by the base catalyzed condensation of diethanolamine and the methyl ester of long chain fatty acids are also important cosmetic ingredients which are nitrosamine susceptible. We have previously reported (11) on a method for the analysis of NDEIA in one of the most widely used alkanolamides: Lauramide DEA. The method involved direct analysis by HPLC using a variable wavelength detector. We now wish to report certain modifications of that method which have allowed us to increase the sensitivity and reliability of the HPLC analysis. First, we have found that the Whatman HPLC column cited in the experimental section gave increased resolution in the during region of NDE1A. Second, we have incorporated a solvent programming procedure for cleaning the column between NDEIA AREA C•LLECTION AREA INJECTION POINT Figure 2. Cocamide DEA spiked with 5.35 ppm of NDEIA, superimposed on an unspiked sample.

248 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS analyses. This has eliminated ghosting which can appear from run to run. Third, detection is accomplished using a Waters fixed wavelength detector, giving greater sensitivity. Fourth, the sample size has been decreased to a 25% solution thus eliminating overloading of the column and lastly, the mobile phase has been changed to 40/60 methanol/water (V/V) with a 25-/al injection. The detection limit for this method is about 200 ppb. Statistical data of the method using 32 observations gave a correlation coefficient (r) of 0.999, with a standard deviation (tr) of +0.361 ng (+0.013 ppm) and a 95% confidence level limit of +0.737 ng (+0.029 ppm). When the reverse phase liquid chromatography method was extended to the analysis of NDEIA in cocamide DEA, it was difficult to separate NDE1A from other early eluting materials, resulting in an unacceptable high value for the detection limit. Also, the aqueous solvent used for the separation of NDE1A from the amides required evaporation prior to silylation, which prolonged the identification procedure using GC/MS confirmation. In an attempt to alleviate both problems, a method was developed to isolate the NDE1A from the amide by normal phase HPLC, followed by either quantitation on the reverse phase system or identification by GC/MS. The cocamide DEA was dissolved in 95/5 chloroform/methanol. The initial separation of NDEIA from the cocamide DEA was accomplished by directly injecting the sample on a /aPorasil column with 95/5 chloroform/methanol as the mobile phase. The collected area was evaporated and redissolved in water followed by reinjection and quantitation on a/aBondapak Q8 column. The evaporated collection from the/aPorasil column was also silylated and the mass spectra was found to be consistent with that of a known spectra of silylated NDEIA (11). Figure 2 shows the chromatogram of a spiked sample of cocamide DEA used to determine the retention time of NDEIA, along with the chromatogram of an unspiked sample, with the collected area indicated. Figure 3 shows the chromatograms of the area collections of the spiked and unspiked samples on the/aBqndapak C18 column. As can be seen in Figure 2, NDEIA could not be detected in the unspiked sample using normal phase chromatography while the collected-reinjected sample (Figure 3) showed a quantitatable peak at 260 ppb. NDEIA standards were collected from a /aPorasil column and the collections were reinjected on a /aBondapak (Qs) column. A statistical analysis using 28 observations was run on the reinjected standards. The correlation coefficient (r) was 0.826 with a standard deviation of + 1.271 ng (+0.051 ppm) and a 95% confidence level limit of +2.618 ng (+0.104 ppm). At this point, we feel the method is acceptable as a screen for NDEIA at 1 ppm or greater. As previously reported in this paper, NDEIA analysis was performed on a/aBondapak column. Since the NDEIA elutes very quickly, care must be taken to wash the column with methanol to prevent subsequent interferences from built-up material. To minimize the column purging time, and increase resolution, a Radial Compression Separation System was substituted for the stainless steel columns. The cocamide DEA sample procedure for NDEIA was followed by first separating the NDE1A on a Radial Pak B cartridge followed by quantitation on a Radial Pak A cartridge. Figure 4 shows the separation of NDEIA from cocamide DEA on the Radial Pak A cartridge (spiked and unspiked). The Radial Pak A cartridge retains NDEIA longer than it is retained on