3048
Anal. Chem. 1986, 58, 3048-3051
Quantitative Extraction and Concentration of Synthetic Water-Soluble Acid Dyes from Aqueous Media Using a Quinine-Chloroform Solution Fumiko Kobayashi, Naoki Ozawa, Junji Hanai, Masakazu Isobe,’ and Tadashi Watabe* Laboratory of Drug Metabolism and Toxicology, Department of Hygienic Chemistry, Tokyo College of Pharmacy, Horinouclii, Hachioji-shi, Tokyo 192-03, J a p a n
Twenty-one water-soluble acid dyes, including eleven azo, five triphenylmethane, four xanthene, and one naphthol derivatives, used at practical concentrations for food coloration, were quantitatively extracted from water and various carbonated beverages into a 0.1 M quinine-chloroform solutlon in the presence of 0.5 M boric acid by brief shaking. Ouantitative extraction of these dyes was also accomplished by the 0.1 M quinine-chloroform solution made conveniently from chloroform, quinine hydrochloride, and sodium hydroxide added successively to water or beverages containing boric acid. Oulnine acted as a countercation on the dyes havlng sulfonic and/or carboxylic acld group(s) to form chloroformsoluble ion-pair complexes. The diacidic base alkaloid interacted with each acid group of mono-, di-, tri-, and tetrasulfonic acid dyes approximately In the ratio 0.8-0.9 to 1. The dyes in the chloroform solution were quantitatively concentrated into a small volume of a sodlum hydroxide solution also by brief shaklng. The convenlent quinine-chloroform method was applicable to the quantitative extraction of a mixture of 12 dyes from carbonated beverages, which are all currently used for food coloration. A high-pressure liquid chromatographic method is also presented for the systematic separation and determinatlon of these 12 dyes following their concentration into the aqueous alkaline solution. The chromatogram was monitored by double-wavelength absorptiometry in the visible and ultraviolet ray regions.
Increasing attention has recently been given to the quantitative monitoring of various synthetic water-soluble acid dyes added to food for their suspected toxicity. However, few rapid and quantitative methods are available for allowing the dyes used a t very diluted concentrations to be simultaneously and highly specifically extracted from food and concentrated for their subsequently direct analysis by HPLC. At the present time, not only in the United States but also in other developed countries, a few more or less than 10 synthetic acid dyes remain permitted in law to use as food-coloring agents after many others have been banned in the last 2 decades for their toxicities (1-3), including carcinogenicity (3). Tetra-n-alkylammonium halides, especially tetra-n-alkylammonium (TBA) bromide (4), have been used for the rapid extraction of the strong dye anions as hydrophobic ion-pair complexes from food with organic solvents since cetyltrimethylammonium bromide was first introduced by Sohgr ( 5 ) for the extraction of six food-coloring agents. However, the tetraalkylammonium cations interact so tightly with various kinds of naturally occurring and artificially added organic anions in food, as well as with dye anions, that miscellaneous acidic materials are extracted into the organic phase from aqueous media, interfering with the subsequent determination Present address: Faculty of Pharmaceutical Sciences,University of Setsunan. 45-1 Nagaotoge-cho. Hirakata-shi, Osaka 537-01, Japan.
of the dyes by HPLC. Moreover, the most widely used, strongly anionic dyes with sulfonic acid groups are very hard to dissociate from the hydrophobic TBA complexes, so a high concentration of perchloric acid may be needed for recovery of the dyes from the ion-pair complexes in the organic phase into the aqueous phase a t higher concentrations (4). The treatment of the TBA-dye complexes with perchloric acid not only results in the low recovery of some of the extracted sulfonic acid dyes to be concentrated in the acidic aqueous phase (4) but also leads to the facile decomposition (fading) of important acid dyes with xanthene structures. Therefore, it is practically difficult to apply the TBA method to the rapid determination by HPLC of a wide variety of water-soluble acid dyes used as dye mixtures a t very low concentrations for food. The weaker base, tri-n-octylamine (TOA), is a recently proposed cationic candidate used at weakly acidic pH for the more specific extraction and concentration of the synthetic water-soluble acid dyes from food (6-9). Actually, the use of TOA may remarkably reduce various organic acids extracted into the organic phase from food. However, the TOA method also needs perchloric acid for the complete dissociation of the extracted acid dyes from the TOA complexes in the organic phase, so it can not be applied to the acid-unstable xanthene dyes (7-9). Aqueous alkaline solutions, including caustic alkali, have been demonstrated not to be effective in dissociating the acid dyes from the TBA- and TOA-dye complexes (6, 7). As has been briefly reported (IO),quinine is one of the most suitable countercation donors among a variety of basic tertiary amines examined for the quantitative extraction of watersoluble, synthetic acid dyes from aqueous solutions. In addition, the hydrophobic quinine-dye complexes formed can be readily dissociated by brief shaking of the organic phase with a very small volume of a low concentration of a caustic alkaline solution, and the water-soluble dyes are quantitatively concentrated into the aqueous alkaline phase. The present paper deals with the method for the rapid and quantitative extraction of very low concentrations of various types of 21 synthetic, water-soluble dyes from aqueous solutions into a quinine-chloroform solution, followed by their quantitative recovery into an aqueous alkaline solution and also with the tentative application of the method to the recovery of a mixture of currently used water-soluble dyes added to various carbonated, nonalcoholic beverages.
EXPERIMENTAL SECTION Materials. The azo dyes, Allura Red AC (C.I. 16035; Food Red 17), Amaranth (C.I. 16185; Food Red 9j, Azo Rubine (C.I. 14720;Food Red 3), New Coccine (C.I. 16255;Food Red 71, Orange I (C.I. 14600; Acid Orange 201, Ponceau R (C.I. 16150; Food Red 5j, Ponceau 3R (C.I. 16155; Food Red 6), Ponceau 6R (C.I. 16290; Food Red 8), Ponceau SX (C.I. 14700;Food Red l j , Sunset Yellow FCF (C.I. 15985;Food Yellow 3), and Tartrazine (C.I. 19140;Food Yellow 4), the triphenylmethane dyes, Acid Violet 6B (C.I. 42640; Food Violet 2), Brilliant Blue FCF ((2.1.42090; Food Blue 21, Fast Green FCF (C.I. 42053; Food Green 3), Guinea Green B (C.I. 42085; Food Green l),and Light Green SF Yellowish (C.I. 42095; Food Green 2), the xanthene dyes, Acid Red (C.I. 45100; Acid
0003-2700/86/0358-3048$01.50/0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Red 52), Erythrosine (C.I. 45430; Food Red 141, Phloxine (C.I. 45410; Acid Red 92), and Rose Bengal (C.I. 45440; Acid Red 94), and the other dyes, Indigo Carmine (C.I. 73015; Food Blue 1)and Naphthol Yellow S (C.I. 10316; Food Yellow l),were purchased from Wako Pure Chemical Industries, Ltd., Osaka. Each dye was examined for purity by HPLC and, if necessary, recrystallized from aqueous methanol. Quinine and its monohydrochloride salt, boric acid, and TBA bromide were also obtained from Wako Pure Chemical Industries, Ltd. Quinine N,-oxide ( I I ) , 4-nitronaphthalene-1-sulfonicacid (12),and y-(1-pyreny1)butyricacid (13)were synthesized by the previous methods. Other reagents used were all reagent grade. Noncolored, carbonated soft drinks used for the recovery of food-coloringagents were purchased from Japanese food stores: three US.brands (in abbreviations, names of companies in parentheses) most widely sold in Japan, MRD (P),SPR (C), and SVU (P), and three Japanese brands, KRL (K), STA (S), and MTC (A). Extraction of Water-Soluble Acid Dyes. For the determination of recoveries of the water-soluble dyes as hydrophobic ion-pair complexes with quinine, aqueous solutions containing various concentrations of each dye and boric acid were mechanically shaken for 10 min with an equal volume of chloroform containing various concentrationsof the free alkaloid. The organic phase separated by centrifugation at 2500 rpm for 10 rnin was then mechanically shaken for 10 min with 0.2-1 vol of 0.1 N NaOH to extract the dye as a sodium salt by dissociating the dye-quinine complex. The aqueous alkaline phase separated by centrifugation under the aforementioned condition was subjected to absorptiometry at the wavelength corresponding to the absorption maximum of each dye in the visible ray region. Recoveries of the dyes from beverages with 0.1-0.5 M free quinine-chloroform solutions were examined in the same manner as described above. Carbonated beverages were heated for 5 min on a boiling water bath to remove carbonic acid before dissolution of the dyes and boric acid (0.5 M as a final concentration). As a matter of practical convenience, the quinine-chloroform method could be modified by using aqueous solutions of quinine hydrochloride and less equivalent sodium hydroxide and chloroform instead of the free quinine-chloroform solution when applied to practical analysis of the dyes in beverages. Percent recoveries of a mixture of 12 dyes from beverages were obtained as follows: to a sample solution (20 mL) were added boric acid (0.8 g), 0.12 M quinine monohydrochloride (5 mL), chloroform (5 mL), and 0.5 M NaOH (1mL) to liberate the free alkaloid. The mixture was mechanically shaken for 10 min. The aqueous layer separated by centrifugation at 2500 rpm for 10 rnin was removed by aspiration. The remaining organic phase was pipetted (4 mL) and shaken with an appropriate volume of 0.5 N NaOH (0.2-0.5 mL) for 10 min. The aqueous alkaline phase separated by centrifugation at 2500 rpm for 10 min was neutralized with an equal volume of 0.5 N H,SO, containing an appropriate internal reference for subsequent HPLC analysis. A higher concentration (0.5 N) of sodium hydroxide was favorable to facilitate the dissociation of the dye-quinine complexes in chloroform when the aqueous alkaline phase used for concentration of the dyes was less than 0.2 vol to the organic phase. High-pressure Liquid Chromatography (HPLC). An Atto Model Constametric I1 high-pressure liquid chromatograph equipped with a Nucleosil 7CI8ODS column (7.5 Wm in particle size, 3.9 mm X 30 cm) and two linearly arranged detectors, one of which was a Shimadzu Model SPD-1 spectrophotometer for recording the absorbancies of the dye peaks at a wavelength of the visible ray region or their spectra in both ultraviolet and visible ray regions and the other a JASCO Model UVIDEC-100 detector for recording their absorbanciesat a wavelength of the ultraviolet ray region, were used. TBA bromide was dissolved in 0.1 M phosphate buffer, pH 7.4, containing an equal molar ratio of sodium hydroxide to generate TBA hydroxide, and diluted with the buffer and methanol for preparing developing solvents.
RESULTS AND DISCUSSION Quantitative Extraction of Water-Soluble Acid Dyes from Aqueous Solutions into Quinine-Chloroform. The alkaloid quinine acted as a countercation donor on watersoluble acid dyes to form hydrophobic ion pairs at weakly
B
Concentration oi HIBOI
3049
C
(M)
Figure 1. Effect of boric acid concentrations on the extraction of various types of sulfonic acid azo dyes with quinine into chloroform. (0) Mono- (Orange I), (0) di- (Sunset Yellow FCF), (0)tri- (Amaranth), and (W) tetra- (Ponceau 6R) sulfonic acid dyes were extracted with a 5 mM quinine-chloroform solution. Concentrations of the dyes used were (A) 0.1 mM, (9)0.01 mM, and (C) 0.005 mM.
acidic pH. The dyes (0.005-0.1 mM) with mono- (Orange I), di- (Sunset Yellow FCF),tri- (Amaranth), and tetra- (Ponceau 6R) sulfonic acid groups were all extracted with a chloroform solution of 5 mM free quinine in higher ratios from water containing a higher concentration of boric acid than from any of various tested buffer solutions, p H 4.0-7.0. The optimum concentration of boric acid was higher than 0.4 M for the extraction of these dyes (0.1 mM) from water with the 5 mM quinine-chloroform solution (Figure 1). When the concentration of quinine in chloroform was increased up to 0.1 M, they were all quantitatively extracted into chloroform even when the concentration of boric acid was decreased to 0.1 M. Chloroform was the most suitable among the tested solvents, such as carbon tetrachloride, methylene dichloride, benzene, toluene, isopropyl ether, ethyl acetate, and isobutyl alcohol, for the quantitative recovery of all 22 dyes, including the aforementioned 4 sulfonic acid dyes, from water containing boric acid. Methylene dichloride was next to chloroform, but the others were unsuitable for this purpose. Quinine, a diacidic base with very different pK, values, 5.07 and 9.7 for the quinoline and quinuclidine moieties, respectively, is likely to interact with the acid dyes in the molar ratios 0.8:l per each acid group for the mono-, di-, and trisulfonic acid dyes and 0.9:l for the tetrasulfonic acid one. The dyes (0.1 m M each) were extracted into chloroform proportional to the amounts of quinine used at lower concentrations (0.5-3.2 mM) in the presence of 0.5 M boric acid. A linear relationship was observed with each dye between logarithmic values of the dye concentrations in the organic to aqueous phases and those of the quinine concentrations (Figure 2), where [DQ],,, and [&Iorp represent the concentrations of the dye-quinine complex and free quinine remaining unbound in the organic phase, respectively, and [DIwater the concentrations of the dyes remaining unextracted in the aqueous phase. The slopes (X) of the lines for dye recovery were 0.8, 1.6, 2.4, and 3.6 for the mono-, di-, tri-, and tetrabasic acid dyes. This strongly suggests that the mono-, di-, tri-, and tetrasulfonic acid dyes would form hydrophobic ion pairs with the diacidic counter base in the molar ratios k0.8, 1:1.6, 1:2.4, and 1:3.6, respectively. Quinine Nl-oxide (2-10 mM), a weak monoacidic base with polarity higher than quinine, was examined for the extraction of the monosulfonic acid dye Orange I (0.1 mM) into chloroform in the presence of 0.5 M boric acid, in order to confirm whether the quinoline moiety of the quinine molecule played a partial role as a counterion donor in the ion-pair complex formation with the acid dye. With respect to the polarity of the N-oxide, it showed a relative retention time of 0.37 to that of quinine (16.5 min) by reverse partition HPLC carried out on the Nucleosil 7C18ODS column in methanol-water (17:3, v/v; 1mL/min). Orange I was extracted into chloroform with linearity (slope, X = 1.0 in the log ([DQ],,,/[DI,,,,r):log [$Iorg graph) a t various concentrations of the N-oxide (Figure 3).
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table I. Extraction of Various Acid Dyes with Quinine from Aqueous Phase into Chloroform
0.0
1
acid dye
*.-.-e Disulfonate x = 1.6 0
Trisulfonate x = 2.4 ....a Tetrasulfonate x = 3.6
0-0
'I
i I
d
-0.5 -0.5
0.0
0.5
(mM)
IogCQl
Figure 2. Relationship between concentrations of quinine and extraction of mono-, di-, tri-, and tetrabasic sulfonic acid azo dyes from aqueous phase into chloroform. Orange I, Sunset Yellow FCF, Amaranth, and Ponceau 6R (0.1 mM each in 0.5 M boric acid) were used as mono-, di-, tri-, and tetrasulfonic acid dyes, respectively.
,. OCH,
Orange I -.-, 0.0
0:5 logC01.,.
Quinine N,-oxide
1.o
(mM)
Figure 3. Relationship between concentrations of quinine N,-oxide and extraction of Orange I from an aqueous phase into chloroform. The concentrations of the dye and boric acid used were 0.1 mM and 0.5 M, respectively.
This strongly suggests that the dye interacted with the N-oxide base in an equimolar ratio. However, as expected from its higher polarity and weaker basicity, quinine N,-oxide could extract a much smaller amount of the acid dye than its mother base. Quinine, used at 0.1 M, was sufficient to extract various water-soluble acid dyes quantitatively into the organic phase from their diluted aqueous solutions, which corresponded to the practical concentrations (0.005-0.1 mM) for commercial beverages. In Table I are listed typical data for the percent recoveries of 22 water-soluble synthetic acid dyes (0.005,0.01, and 0.1 mM each), including 11 azo, 5 triphenylmethane, 4 xanthene, and 2 other derivatives, which were dissolved in water containing 0.5 M boric acid. The percent recoveries of all the dyes used, except Indigo Carmine, were quantitative a t each concentration as far as carried out by using a 0.1 M quinine-chloroform solution. When the concentration of quinine was increased to 0.5 M, the recovery of Indigo Carmine from the aqueous phase increased up to 97% without any influence on the quantitative recoveries of the other 21 dyes. Application of the QuinineChloroform Method to the Extraction of Synthetic Acid Dyes from Beverages. Each of the 22 water-soluble synthetic dyes was added to six commercial brands of noncolored, nonalcoholic, carbonated beverages containing various flavoring agents and extracted with
azo Allura Red AC Amaranth Azo Rubine New Coccine Orange I Ponceau R Ponceau 3R Ponceau 6R Ponceau SX Sunset Yellow FCF Tartrazine triphenylmethane Acid Violet 6B Brilliant Blue FCF Fast Green FCF Guinea Green B Light Green SF Yellowish xanthene Acid Red Erythrosine Phloxine Rose Bengal other Indigo Carmineb Naphthol Yellow S
no. and type of acid group %SO33-SO32-S033-SOc l-SO3'&SO32-SOf
70 extracted into CHCl," at the given concns of dye 0.005 0.01 0.1 mM mM mM
100.0 100.0 100.0 100.0
100.2 100.0 100.0 100.0 101.1 100.2 100.0
100.0 100.0 99.9 101.0 100.0 100.0 101.0
100.0
100.0 100.0
2-S03%SO3-
100.0 100.0 101.1 100.0 101.0 100.2
%SO3-, 1-COO-
100.0
100.0
99.1
24033-SO3-
100.0
100.0
100.0 101.0
100.0 99.7
3-SO;
100.0
100.0
99.8
2-so3
101.0
100.0
100.0
3-SO3
101.0
100.0
99.6
2-S03-
100.0 100.7 100.0 100.5
100.0 100.9 100.6
99.9 100.0 100.0 101.0
%SO3-
76.8
73.9
74.2
1-SO3
100.6
100.0
100.1
4-SOL
1-coo1-coo1-coo-
100.0 100.0
100.0
100.0
'The dyes were extracted with a 0.1 M quinine-chloroform solution from equal volumes of 0.5 M boric acid solutions, reextracted into 0.2-1 vol of 0.1 N NaOH, and measured by absorptiometry at wavelengths for their peak maxima. Data are expressed as arithmetic mean values of at least five experiments. *The dye, unstable to 0.1 N NaOH, was quantitatively reextracted with 0.1 M Na2CO3-NaHCO3buffer, pH 10.0, from the organic phase. In this buffer solution, the dye was stable enough t o remain unchanged in its absorbancy for at least 90 min. the 0.1 M free quinine-chloroform solution in the presence of boric acid (0.5 M) under the same conditions as used for the recovery of the dye from water. The percent recoveries of the dye (0.005-0.1 mM) from the six beverages did not show any appreciable difference from those from water shown in Table I. There was also no appreciable difference in recovery of the aforementioned concentrations of each dye from the carbonated beverages when the 0.1 M free quinine-chloroform solution was replaced by that prepared from chloroform, quinine hydrochloride, and less equivalent sodium hydroxide added successively to the samples. Of the tested 22 dyes were chosen 12, all currently used for food coloration, consisting of 6 azo, 2 triphenylmethane, and 4 xanthene derivatives, and they were added as a mixture to the carbonated beverages in order to confirm whether the dye mixture used at practical concentrations ( 5 and 50 fig each/mL) could be simultaneously extracted with the 0.1 M quinine-chloroform solution and concentrated in as small a volume of the caustic alkaline solution as possible. All the colors of the dye mixture in the commercial beverages were completely extracted in the presence of boric acid (0.5 M) into chloroform containing 0.1 M quinine liberated from quinine
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table 111. Recovery of Acid Dyes Added to a Colorless Carbonated Beverage by the Quinine-HPLC Method
Table 11. Analytical Data for Separation and Determination of Acid Dyes by HPLC-Absorptiometry
dye
ta
at 255 nm at 510 nm azo 500 20 800 Tartrazine 23600 17400 Amaranth 12900 20700 Sunset Yellow FCF 23100 20700 New Coccine 13100 20800 Allura Red AC 5820 23000 Azo Rubine triphenylmethane at 310 nrn at 625 nm 14000 98300 Fast Green FCF 13500 101100 Brilliant Blue FCF at 260 nm at 545 nm xanthene 25200 46500 Acid Red 29500 42200 Erythrosine 22400 91300 Phloxine 36200 56600 Rose Bengal
solvent systemb
retention time: rnin
A A A
3.1 3.7 6.0
A A A
7.8 15.3 39.3
B B
10.9 15.0
C C C C
3051
6.7 8.6
23.4 36.4
Data obtained in the developing solvents for HPLC. *Column, Nucleosil 7C18(7.5 wm in particle size, 3.9 mm X 30 cm); column temperature, 30 "C; developing solvents contained 1 mM TBA and consisted of MeOH and 0.1 M phosphate buffer, pH 7.4, in the ratios 4:8 (A), 9:11 (B), and 31:19 (C); flow rate, 1 mL/min; internal standards, p-chlorobenzoic acid (6.8 min) in solvent A, 4nitronaphthalene-1-sulfonicacid (6.7 min) in solvent B, and y(1pyreny1)butyric acid (15.9 min) in solvent C. CDataare expressed as arithmetic mean values of at least three experiments. hydrochloride and sodium hydroxide. T h e dyes existing as complexes with quinine in the organic phase were then quantitatively reextracted into 1/20 to 1/4 vol of 0.5 N NaOH. It was favorable t o use a higher concentration (0.5 N) of sodium hydroxide for facilitating the dissociation of the dye-quinine complexes on decreasing the voluminal ratio of the aqueous to organic phases to less than 0.2. The aqueous alkaline solution containing sodium salts of the dyes was neutralized and analyzed by HPLC on t h e ODS column in methanol-phosphate buffer, p H 7.4, containing TBA as a countercation. The three groups of the dyes showed a marked difference in retention time group by group (Table 11). The azo dyes were the most polar with the shortest retention times and the xanthenes the most nonpolar under the HPLC conditions. A combination of UV absorptiometry and colorimetry was suitable for the specific detection and determination of all the dyes in each group in the chromatograms. Absorbancy at 255 and 510 nm, 310 and 625 nm, and 260 and 545 n m were suitable for azo, triphenylmethane, and xanthene dyes, respectively. Typical data for the recovery of the 12 dyes from the commercial beverage SPR (C) are shown in Table 111. All the dyes added were recovered almost quantitatively (higher than 92%). Very similar results were also obtained with the other five commercial carbonated drinks examined. The present investigation provides a rapid method for the quantitative extraction of very low Concentrations of synthetic water-soluble acid dyes from commercial carbonated drinks as well as from aqueous solutions. Simultaneous analysis of dye mixtures has been achieved,with high accuracy by combining the quinine-chloroform extraction method and HPLC. It should be emphasized that the dyes in beverages even at a concentration of 0.4 wg/mL can be determined by reducing the volume of the caustic alkaline solution used for extracting them as sodium salts from the quinine-chloroform solution. Sodium hydroxide, however, could not be used for the dissociation of the alkali-unstable acid dye Indigo Carmine from the quinine complex because of its facile decomposition under
recovery,a 70,at given concns azo Allura Red AC Amaranth Azo Rubine New Coccine Sunset Yellow FCF Tartrazine triphenylmethane Brilliant Blue FCF Fast Green FCF xanthene Acid Red Erythrosine Phloxine Rose Bengal
96.0 96.5 96.5 93.5 95.5 97.3
95.5 97.7 93.5 92.2 99.8 97.4
00.0
97.7
95.7 97.5
99.7 98.6 96.3 100.0
98.4 97.6 93.0 101.0
DThedyes extracted in the presence of 0.5 M boric acid with chloroform containing 0.1 M quinine liberated from its hydrochloride with NaOH were concentrated into 0.5 N NaOH and determined by HPLC as described in Table I1 following immediate neutralization with 0.5 N H2S04. Data are expressed as arithmetic mean values of at least three experiments. the alkaline condition used. Instead, a carbonate buffer, p H 10.0, was effective in extracting the dye without decomposition from the chloroform solution into the aqueous phase. Application of the quinine-chloroform method to the extraction of synthetic water-soluble acid dyes added to food other than beverages is now in progress in our laboratory. The present method, combined with a simple pretreatment, was found to be useful for the rapid, highly specific, and quantitative extraction and concentration of a wide variety of the synthetic water-soluble acid dyes from solid foods. These data will be published elsewhere. Registry No. C.I. 16035,25956-17-6; C.I. 16185,915-67-3; C.I. 14720, 3567-69-9; C.I. 16255,2611-82-7;C.I. 14600, 523-44-4; C.I. 16150,3761-53-3; C.I. 16155,3564-09-8; C.I. 16290, 5850-44-2; C.I. 14700,4548-53-2;C.I. 15985,2783-94-0; C.I. 19140,1934-21-0; C.I. 42640, 1694-09-3;C.I. 42090,3844-45-9; C.I. 42053, 2353-45-9; C.I. 42085,4680-78-8; C.I. 42095, 5141-20-8;C.I. 45100,3520-42-1; C.I. 45430, 16423-68-0; '2.1. 45410, 18472-87-2; C.I. 45440, 632-68-8; C.I. 73015,860-22-0;C.I. 10316,846-70-8;quinine, 130-95-0;quinine "oxide, 70116-00-6.
LITERATURE CITED (1) Hansen, W. H.; Davis, K. J.; Fitzhugh, 0. G.; Nelson, A. A. Toxicol. Appl. Pharmacol. 1983, 5 , 105-118. (2) Haveland-Smith, R. B.; Combe, R. D. Food Cosmet. Toxicoi. 1980, 18, 215-221. (3) Noonan, J. E.; Meggos, H. CRC Handbook of Food Additives, 2nd ed.; Furia, T. E., Ed.; CRC: Boca Raton, FL, 1980; Vol. 2, pp 339-383. (4) Masiala-Tsobo,C. Anal. Lett. 1980, 13, 985-1000. (5) Sohir, J. Z . Lebensm. Unters. Forsch. 1987, 132, 359-362. (6) Puttemans, M. L.; Dryon, L.; Massart, D. L. Anal. Chim. Acta 1980, 113, 307-313. (7) Puttemans, M. L.; Dryon, L.; Massart, D. L. J . Assoc. Off. Anal. Chem. 1982, 6 5 , 737-744. (8) Puttemans, M. L.; Dryon, L.; Massart, D. L. J . Assoc. Off. Anal. Chem. 1983, 6 6 , 1039-1044. (9) Puttemans, M. L.; Voogt, M.; Dryon, L.; Massart, D.L. J . Assoc. Off. Anal. Chem. 1985, 6 8 , 143-145. (10) Kobayashi, F.; Isobe, M.; Watabe, T . Proceedings of the 99th Annual Meeting of Pharmaceutical Sociery of Japan, 1979; p 378. (11) Ochiai, E.; Kobayashi, G.; Hasegawa, K. Yakugaku Zasshi 1947, 6 7 , 101-103. (12) Woroshzow, N. N.; Koslow. W. W.; Trawkin, I . S. Chem. Z . 1940, I , 690. (13) Bachman,W. E.; Carmack, M.; Safir, S.R. J . Am. Chem. SOC.1941, 6 3 , 1682-1685.
RECEIVED for review March 4, 1986. Accepted July 23, 1986. Presented at the 99th Annual Meeting of Pharmaceutical Society of Japan, in Sapporo, 1979.
