J. Agric. Food Chem. 1888, 36, 1307-1309
1307
Total Residues of 2-Naphthoxyacetic Acid, 2-(6-Hydroxynaphthoxy)acetic Acid, and 2-(7-Hydroxynaphthoxy)acetic Acid in Tomato Plants and Fruit by High-Performance Liquid Chromatography Thomas E. Archer* and James D. Stokes
A study was undertaken to determine the total residues including the sugar- and protein-bound residues of 2-naphthoxyacetic acid (BNOA), 2-(6-hydroxynaphthoxy)aceticacid (6-OH-BNOA), and 247hydroxynaphth0xy)acetic acid (7-OH-BNOA) in tomato fruit, blossoms, and leaves from plants treated with BNOA. Analyses were by high-performance liquid chromatography. There were no detectable residues of 7-OH-BNOA a t a method sensitivity of 0.05 ppm on any of the samples and C0.05 ppm of BNOA and 6-OH-BNOA on the tomato fruit. Tomato plant leaves had small amounts of BNOA and 6-OH-BNOA.
Various chemicals of the plant growth regulating hormone types such as BNOA, 4-chlorophenoxyacetic acid, and 2,4-dichlorophenoxyaceticacid have been investigated for their effects on tomatoes (Mann and Minges, 1949; Wittwer and Reath, 1952). Emphasis in these studies was on tomatoes grown for early market production rather than on those grown for processing. Rathore et al. (1983) separated standard mixtures of organic acids including 2-naphthoxyacetic acid (BNOA) by paper chromatography. Thin-layer chromatography has also been used for separations of similar compounds (Gupta et al., 1984; Rathore et al., 1985). Baldi et al. (1985) homogenized apples and tomatoes in acidified acetone for extraction and analyzed 1-naphthaleneacetic acid and BNOA as their pentatluorobenzyl derivatives by gas-liquid chromatography. Fechner (1981) analyzed BNOA as its methyl ester and 2-naphthol after separation by highperformance liquid chromatography. Archer and Stokes (1980) determined the levels of BNOA and 2-naphthol on field-sprayed tomato blossoms, leaves, and fruits by high-performance liquid chromatography and an ultraviolet light detector. An inclusive computer search of the literature from 1967 to 1986 did not show any information about the two possible metabolites of 2-naphthoxyacetic acid (BNOA), the plant growth regulator, namely 2-(6-hydroxynaphthoxy)acetic acid (6-OH-BNOA) and 247-hydroxynaphthoxy)acetic acid (7-OH-BNOA) (CAS, STN, 1967-1986). The present study was undertaken to determine the total residues including the sugar- and protein-bound residues of BNOA, 6-OH-BNOA, and 7-OH-BNOA in tomato fruit, blossoms, and leaves from plants treated with BNOA. No reports are in the current literature concerning the detection or levels of 6-OH-BNOA or 7-OH-BNOA, which are possible metabolites of BNOA when applied to materials such as tomato blossoms or plants. MATERIALS AND METHODS Reagents. Pesticide-grade acetone and acetonitrile, reagent-grade anhydrous ethyl ether, and HPLC-grade methanol were the purified solvents used. Silica gel, grade 923, was used as received from Davison Chemicals, Baltimore, MD. BNOA was provided by Chevron Chemical, Ortho Division, San Francisco, CA. 6-OH-BNOA and 7-OH-BNOA Department of Environmental Toxicology, University of California, Davis, California 95616. 0021-8561/88/1436-1307$01.50/0
were prepared by reacting equimolar amounts of the corresponding naphthalenediol with chloroacetic acid, and they were purified by liquid chromatography. The 2,6-naphthalenediol was acquired from Aldrich and purified in the following manner: A mixture of 5.0 g of technical-grade material, 5.8 mL of acetic acid anhydride, and 8.4 g of sodium acetate was heated under reflux for 3 h. While still warm, the mixture was poured into 100 mL of water with stirring. The precipitate that formed, after drying in air, weighed 6.73 g (88%); mp 172-174 "C [lit. (Chatt and Wynne, 1943) mp 173 "C]. The crude 2,6-diacetoxynaphthalenewas recrystallized from ethermethylene chloride to give 4.14 g of shimmering white needles, mp 176177.5 "C. A mixture of 50 mL of degassed (N,) 1 N aqueous sodium hydroxide and 2.02 g of the diester was heated with hot water until it became homogeneous. About 30 min was required. The solution was cooled and acidified tQpH 5.3 with 3 N hydrochloric acid to precipitate the diol. The yield of dried product was 1.07 g; mp 222-223 "C [lit. mp 222 "C]. 6-OH-BNOA was prepared by adding 0.78 g of purified 2,6-naphthalenediol to 4.0 mL of 5.0 N aqueous sodium hydroxide that had been well flushed with nitrogen. The mixture, under nitrogen, was stirred and heated in a boiling water bath for about 10 min to dissolve the diol, and 0.46 g of chloroacetic acid was added in one portion. Heating and stirring were continued for 45 min, during which time a small amount of precipitate formed. After the mixture was allowed to cool to room temperature, it was acidified with 6 N hydrochloric acid and stirred for an additional 10 min to coprecipitate the product and untreated diol. When dry, the light brown solid weighed 0.85 g. A portion was dissolved in 50% aqueous methanol containing 0.1% phosphoric acid and analyzed by HPLC on a Whatman Partisil M9 ODS 3 column with a flow rate of 6.0 mL/min. At 260 nm, relative responses of 43, 51, and 5 were observed for the diol, 6-OH-BNOA, and 2,6-naphthalenedioxyacetic acid. TLC (SiOz, acetone) gave R values of 0.68, 0.37, and 0.03 for the diol, 6-OH-BNOA, and the diacid. Column chromatography (SiO,, acetone) of a 170-mg portion of the crude product gave 78 mg of 6-OHBNOA as a light gray solid, mp 164-166 "C [lit. (Byrde et al., 1956) mp 165-166 "C]. 'H NMR (360 MHz, acetoned,): 6 4.8 (s, CH,), 7.11 (d, J = 9 Hz,C3-H or C,-H), 7.21 (br s, C1-H or C,-H), 7.62 (d, J = 8.0 Hz, C4-H or C8-H),7.66 (d, J = 10.09 Hz, C4-H or C8-H). MS (70 eV) m / z (re1 intens): 218 [MI+ (100),160 (ClJ-I8OZ)'(32), 159 (Ci&@z)+(91), 143 (CloH70)' (121, 131 (CgH70)' (go), @ 1988 American Chemical Society
1308 J. Agric. F O O ~Chem., Voi. 36,No. 6, 1988
Archer and Stokes
Table I. Residues of BNOA and 6-OH-BNOA on Field-Treated Tomato Leaves and Blossomsa residues) ppm leaves blossoms size of spray treatment no. fruit, g expt day no. BNOA 6-OH-BNOA BNOA 6-OH-BNOA 1 0 2.40 f 0.18
