SHAMPOO PRESERVATIVE TESTING 309 temperature, 100øF, and 120øF (i.e., 3 ø, 20 ø, 38 ø, and 49øC, respectively)for the duration of the stability study. One bottle of product stored at each temperature was removed at specified times and subjected to preservative efficacy testing. After sam- pling, the bottles were placed in the sample storage archives. TEST PROCEDURE A 0.1-mL aliquot of test organism suspension, containing about 108 organisms/mL, was added to ca. 50=mL portions of each test sample in 100=mL screw-capped bottles. Aerobic plate counts were determined, and D-values were calculated as described in an earlier report (4). HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Test samples of shampoo were diluted to 1% (wt/vol) in mobile phase. After filtration, the samples were assayed by injecting 250-1zL aliquots onto a 250 x 4.6 mm i.d. LiChrosorb © RP-18 (5 Izm) column with a 40 X 4.6 mm i.d. guard column con- taining Perisorb © RP-18, 30-40 Izm. Chromatographic conditions were as follows: mobile phase = water:methanol (45:55) with 0.2% acetic acid, flow rate 0.5 mL/min, temperature 25øC, and detector range 0.05 AUFS. The column effluent was monitored at a UV wavelength of 275 nm. CALCULATION OF APPARENT ACTIVATION ENERGY The apparent activation energy (Ea') for the shampoo preservative system was deter= mined for each test organism at each time period of the stability study (i.e., 1, 3, 6, or 12 mo.). A plot of Log 2.303/D-value vs 1/T (where T is the absolute temperature in øK) was calculated for each test organism, and the slope of each line was determined by linear regression. The Ea' has a value of - 2.303R (slope), which is the same calcula- tion as for Ea (17). Preservative efficacy tests were not performed on samples stored beyond 6 mo. at 49øC or 12 mo. at 38øC. The D-values obtained on samples stored for 18 mo. at 3 ø and 20øC were not used in calculation of Ea' values because D-values at the higher temperatures were not available. RESULTS Figure ! shows the changes in preservative efficacy that occurred during storage of the shampoo at 3 ø, 20 ø, 38 ø, and 49øC for 18 mo. when using E. coli as the challenge organism. It is apparent that the preservative system was not stable and that the D- values increased with increasing storage time and temperature. Thus, the D-values for E. coli changed from •4 hr at the outset of the study to 35 hr after storage for 1 mo. at 49øC. The preservative efficacy decreased more slowly in samples stored at 38øC than at 49øC, and samples stored at 3 ø and 20øC were affected much less than those stored at 38øC, as indicated by smaller changes in D-values during the test. The preservative efficacy test results obtained when P. aeruginosa was inoculated into shampoo samples that had been stored up to 18 mo. at different temperatures are presented in Figure 2. The findings obtained with P. aeruginosa were similar to those

310 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS lOO go 80 70 60 50 4.0 3o 20 lO o i i i i i i i i i [ i i i i i i i 0 2 4 6 8 10 12 14 16 18 MONTHS ON STABIUTY TEST Figure 1. D-values for E. coli in shampoo after storage at 3 ø, 20 ø, 3 , and 49øC for l, 3, 6, 12, and 18 . 8 ø mo. Explanation of symbols: i,__i,, shampoo stored at 49øC O--O, shampoo stored at 3 C I--I, I--•, shampoo stored at 20øC and }--{, shampoo stored at 3øC. obtained with E. coli however, comparison of Figures 1 and 2 reveals that the test strain of E. coli was slightly more resistant to the preservative system than the test strain of P. aeruginosa used in these studies. lOO 90 80 70 60 50 •o 30 20 lO o , 0 2 • 6 i i i i i i i i i 8 10 12 14 16 18 MONTHS ON STABILITY TEST 8 o Figure 2. D-values for P. aeruginosa in shampoo after storage at 3 ø, 20 ø, 3 , and 49øC for 1, 3, 6, 12, and 18 mo. Explanation of symbols: i,__i,, shampoo stored at 49øC O--•, shampoo stored at 38øC •--•, shampoo stored at 20øC and [•--I, shampoo stored at 3øC.