Anal. Chern. 1983, 55, 1229-1232
in that there is a minimum and a maximum (1.25-2 h). If solutions containing moderately large amounts of 2,4,5,6tetrachloroaniline are allowed to stand for more than 2 h, the purplish red dye starts to precipitate. The color obtained with 2,4,5,6-tetrachloroanilineis the least sensitive of all the aromatic amines tested. Accuracy and Precision. The calibration curves for all the aromatic amines followed Beer's law. The accuracy and precision were tested by carrying aliquots of standard solution B through the procedures. The average error for all the aromatic amines except 2,4,5,6-tetrachloroanilinewas 0.0014 was 0.0042 mg; the average error for 2,4,5,6-tetrachloroaniline mg. The average standard deviation for all the aromatic amines ( N = 4) except 2,4,5,6-tetrachloroanilinewas *0.0016 mg; the average standard deviation for 2,4,5,6-tetrachloroaniline was *0.0044 mg.
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Registry No. N-na, 551-09-7; 2-aminophenol, 95-55-6; 3aminophenol, 591-27-5;4-aminophenol, 123-30-8;p-phenylenediamine, 106-50-3;m-phenylenediamine, 108-45-2; 2,4-dinitroaniline, 97-02-9;2,6-dinitroaniline,606-22-4;2,4,5-trichloroaniline, 636-30-6;2,4,6-trichloroaniline,634-93-5;2,4,5,6-tetrachloroaniline, 654-36-4. LITERATURE CITED (1) Norwitz, G.; Keliher, P. N. Anal. Chern. l W 2 , 54, 807-809. (2) Nolier, C. R. "Chemistry of Organic Compounds", 3rd ed.; W. B. Saunders: Phlladelphia, PA, 1965; p 566. (3) Allinger, N. L.; Cava, M. P.; DeJongh, D. C.; Johnson, C. R.; Label, N. A.; Stevens, C. L. "Organic Chemistry"; Worth Publishers: New York, 1972; pp 234-235. (4) Zoliinger, H. "Dlazo and Azo Chemistry"; Interscience: New York, 1961; pp 27-37, 140-141, 152-153, 262-263.
RECEIVED for review September 16, 1982. Accepted April 4, 1983.
Comparison of Methods for Determination of Ascorbic Acid in Animal Tissues Robert Scott Carr,"' Marcel B. Bally,* Peter T h ~ r n a s and , ~ Jerry M. Neff' Deparfrnent of Bioiogy, Texas A&M University, Coliege Station, Texas 77843
Tlssues from a wlde varlety of animals were analyzed for ascorbic acld slmultaneously by several commonly used coiorlmetrlc procedures and a newly developed technique3 utilizing liquid chromatography with electrochemlcal delectlon (LCEC). The colorimetric method which utilizes 2,4-dinltrophenylhydrazine produced values which compared reasonably well wlth the values obtained by LCEC whlle the values determined by the colorlmetrlc procedures whlch use a,a'-bipyrldyl were often slgnlficanlly elevated due to lnterferlng substances. Due to the great specles, tlssue, and assay-dependent variability inherent In tissue ascorblc acld determinatlons, it is recommended that data from past and future studles be evaluated prudently.
Since the antiscorbutic properties of ascorbic acid were first discovered, the physiological role of this vitamin in animal nutrition has been studied extensively. Numerous techniques have been developed to estimate the concentration of ascorbic acid in animal tissues and fluids, the majority of which have utilized colorimetric procedures. Most assays for ascorbic acid are based upon the oxidation-reduction properties of this vitamin. The most commonly used methods are modifications of the colorimetric techniques of either Roe and Kuether (1)or Sullivan and Clarke (2). Roe and Kuether used 2,6-dichlorophenol-indophenolto oxidize ascorbic acid to dehydroascorbic acid, which reacts with (2,4-dinitrophenyl)hydrazine (DNPM) t o form a hydrazone derivative which can be measured colorimetrically. The Battelle New England Marine Research Laboratory,Washington Street. Duxburv. MA 02332. Departmen; 'of Biochemistry, University of British Columbia, Vancouver, B.C., Canada V6T 1W5. Port Aransas Marine Laboratory,The University of Texas Marine Science Institute, Port Aransas, TX 78373.
procedure of Sullivan and Clarke is based on the reduction of Fe3+ to %e'2+ by ascorbic acid. The Fe2+reacts with a,dbipyridyl to form a complex which can also be determined colorimetrically to estimate the ascorbic acid concentration. Modifications of these two colorimetric techniques were used in the present study (3, 4 ) . Recently a new technique utilizing liquid chromatography with electrochemical detection (LCEC) has been developed to quantify the ascorbic acid content of animal tissues (5). Due to the chromatographic separation and the selectivity of electrochemical detection, this method provides a greater selectivity than the colorimetric procedures. During preliminary investigations to determine the suitability of the aforementioned methods for estimating the ascorbic acid content of various animal tissues, considerable variability was observed among the various techniques. In order to evaluate the reliability of the colorimetric techniques, a series of simultaneous ascorbic acid determinations was made on a variety of vertebrate and invertebrate tissues and plasma.
EXPERIMENTAL SECTION Sample Preparation. All tissues were freshly excised except those of the mullet and the grass shrimp Puluemonetes pugio, which were removed from animals frozen in liquid nitrogen. Preweighed tissues kept on ice were homogenized with a motorized tissue grinder in the deproteinization buffer which was ice-cold 250 mM HC104containing 5% trichloroacetic acid (TCA, 5 1 0 % (w/v)). The homogenates were centrifuged at 27 OOOg for 30 min. The supernatant was transferred to clean test tubes and recentrifuged if particulate matter was present. Plasma or red blood cells obtained from freshly drawn heparinized blood were deproteinized with ice-cold 50% TCA (1vol TCA/10 vol sample) for 30 min at 4 "C. After the samples were centrifuged, the supernatants were decanted and kept on ice until aliquots of each sample were assayed for ascorbic acid. Samples containing high concentrations of ascorbic acid were diluted with the deproteinization buffer in order to obtain a concentration within the linear working range of the assays before the ascorbic acid content was
0003-2700/83/0355-1229$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Table I. Recovery Data for the DNPH, a,a'.Bipyridyl, and LCEC Methods in Wlnich a Known Amount of Ascorbic Acid Was Added to the Tissue Homogenate" % recovery
homogenate (w/v)
DNPH
ethanol solubilized a,a'-bipyridyl
1 2 5 10 15
99 100 98 95 a4
110 104 87 41 15
%
LCEC
ia4
a Recovery experiments were performed with goldfish livers for the DNPH and a,a'-bipyridyl assays and with polychaete posterior segments for the LCEC assay.
determined. Ascorbic acid standards in cold deproteinization buffer were prepared ffresh daily and the same set of standards was used to produce standard curves for each of the methods. DNPH Method. Ascorbic acid was assayed by modification of the technique of Terada et al. (3). Ascorbic acid was oxidized to dehydroascorbic acid by addition of 25 pL of 0.2% 2,6-dichlorophenol-indophenol (2,6-Dye)to 250 pL of deproteinized sample. After a 1h incubation at room temperature, 250-pL of thiourea reagent (2% thiourea in 5% metaphosphoric acid) was added followed by an equal volume of 2% DNPH in 12 M HzS04. The samples were incutbated for 3 h at 60 "C after which 0.5 mL of ice cold 18 M HZSO4was added. The samples were centrifuged at 500g for 10 min and allowed to reach room temperature before the absorbance at 524 inm was determined with a dual beam Pye Unicam SP 1800 spectrophotometerwith deproteinizationbuffer used for the blank. C(ontro1samples contained H 2 0 instead of 2,6-Dye. The concentration of ascorbic acid was determined from the difference between the absorbance reading of the duplicate experimental and control samples. a,d-Bipyridyl Technique. Ascorbic acid was determined with either water or ethanol-solubilizeda,a'-bipyridyl. The following reagents were added in, sequence to 250 pL of the deproteinized samples: 25 pL of 85% 0-H3P04,200 pL of a 1% aqueous solution of a,a'-bipyridyl (solubilized by heating at 60 "C for 5 min), and 25 pL of 3% ferric chloride. In the assay utilizing ethanol solubilized a,a'-bipyridyl, 50 pL of 85% 0-H3P04,30 pL of 8% a,d-bipyridyl in ethanol, and 50 pL of 3% ferric chloride were added to 500 NL of deproteinixed sample. The assay mixture was incubated at room temperature for 30 min and the absorbance at 525 nm was measured against a reagent blank. Determination by LCEC. Ascorbic acid was determined by the procedure of Carr and Neff (5). A Glenco System I HPLC equipped with an amperometric detector (BioanalyticalSystems Inc., Model LC-4) was employed. Ascorbic acid in the deproteinized samples was separated on a 25 cm X 4.6 mm column of Whatman Partisill0 SAX chromatographic resin using 60 mM sodium acetate (pH 4.8) as the mobile phase at a flow rate of 2.9 mL/min. A potential of 750 mV vs. Ag/AgCl was applied to the thin-layer carbon paste electrode. This method is capable of detecting 2 ng of ascorbic acid in a 10-pL sample. The concentration of ascorbic acidl in the samples was determined by comparison with a regression line of peak area vs. concentration for freshly prepared, serially diluted ascorbic acid standards in deproteinizing buffer. The samples were always analyzed within 3 h of homogenization. The oxidation of ascorbic acid in de-
proteinizing buffer at 4 OC during this time period is negligible (
