276 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS CH 3 Red Lake C Amine diazotization CH3 diazonium salt I coupling I CH 3 O• I D&C Red No.8 SO 3Ba/ OH , /2 C N--N 8-naphtho 1 CH • • D&C Red No.9 By the analysis of D&C Red No. 9, eleven subsidiary colors shown in Table I were confirmed. Among these impurities was 1-phenylazo-2-naphthol (P-0) which is reported to be a contact sensitizer. The subsidiary colors are methyl- or chloro-derivatives of the basic skeletal structure (phenylazo-naphthol) of D&C Red No. 9. D&C Red No. 9 is the compound produced by diazotization of Red Lake C Amine and by coupling with Table II Synthesized Aromatic Azo Compounds 3 2 OH Subttltuentl and Starting materials • their position Yiek • 2 3 4 5: 6 Materials for Coupling (g) : dl•zotization compound P-0 R©age•t (Sudan ! ) / P-1 CI o-Chioroaniline •-naphthol 1.0 Po2 CI m-Chloroaniline # 1.1 P-3 Ci )- Chloro•niiine # 0.9 p-4 CH=: Reagent (O•ange SS) / P-5 iCI-b m-Toluidine •-naphl h4d 4• .. .... P-6 CI-b 3- Toiuidine # 5• P-7 CI •.H•' 2-Chioro-3-nilrotoiue•e # 0•5 P-8 CI 'CI-b' 3-Chioro-4-nilrotoiu• # 5.4 P-9 CI. .CH3 $-Chiozo-m-loiuidine # 2.4 P-10 Ci Cl• $-Chiozo-o-toiuidine # 3• Poll •-b i CI 3-Chioro-o-toluidine # 4.0 P-12 ! CI :.CH=i 3-Chloro-p-loluidlne 3•) Po13 'CI: iCI.• 4-Chlo•o-2-nitzoloiue•e # 4-• , .. P-14 .•1•. ! CI 4oChlo•o-o-toluidirte # •.0 P-15 .C•bi CI . 4-Chlotoom-loiuidlrte # 3.9 P-l• CI ' :CI •i Z4-1:Nchloro-5-nltrotoluene ,, 0.4 l•-17 ß CI iCI-b: i CI 2.S.-I:Nchlol•.M-nltrotoluene # 2• F)-18 . CI i CI '• 3.4-D•chlMo-&-nltrotoluene ,, 1:) , , , i

D&C RED NO. 9 ANALYSIS AND PURIFICATION 277 fi-naphthol to give D&C Red No. $, which is converted to the barium salt, D&C Red No. 9. The desulfonation reaction hardly seems possible in this process. Accordingly, it seems likely that the confirmed subsidiary colors are produced from the aromatic amine impurities contained in Red Lake C Amine and fi-naphthol. In order to prepare the standard samples for the quantitative analysis and the contact sensitization test of the subsidiary colors in D&C Red No. 9, nineteen kinds of both subsidiary colors, the structural formulae of which had been confirmed, and related compounds were synthesized (Table II) as standard samples. Each aromatic azo compound was synthesized from the corresponding amine and fi-naphthol. When pertinent aromatic amines were not commercially available, the aromatic nitro- compounds were reduced to aromatic amines which were used for raw materials. The synthesized aromatic azo compounds were purified by recrystallization using acetone, and their structural formulae were confirmed by GC-MS, NMR, etc. The subsidiary colors in various samples of D&C Red No. 9 were quantitatively analyzed. As shown in Figure 4, D&C Red No. 9 was dissolved in 1% (V/V) sulfuric I D&C Red No.9 0.05g I (Weigh exactly) :::::.....:• ( Dissolve with 1% sulfuric acid/DMF, 25ml • Add water, 75ml Pass this solution through SEP-PAK C,8 cartridge using 100ml syringe Wash with 50r•l water Elute with 5m• acetone and then 20ml chloroform •.•Evaporate to iryness with rotary evaporator(•Te-'-•p..'•025•--•m,methanol ( Add 150,--,200/zl 100ml syringe IHPLC-]]]:I Col"•-'•mn TSK LS-41• ":•+-SEP PAK C•8 Eluent : Methanol, 1.5ml/min. • cartridge Detector: VIS (436nm), 0.5AUFS -] r Inj. vol.: ca. 150p. I •) 5 •10ml/min. I I I Fractiøn All Fraction B I I I I IFraction C I IFraction D I I I Evaporate to dryness - Add 1% ethyl acetate/n-hexane. 1.00ml Column : Nucleosil-50*, 4mm i.d.x 25cm, Temp.: 30øC Eluent : 1.0% ethyl acetate/n-hexane, 2.0ml/min. Detector: VIS (436nm), 0.01 AUFS , Macherey-Nagel (GER) Figure 4. Flow chart for determination of subsidiary colors in D&C Red No. 9.