(12) E. Klesper, A. H. Corwin, and D. A. Turner, J. Org. Chem., 27, 700 (1962). (13) S. T. Sie and G. W. A. Rijnders, Sep. Sci., 2, 755 (1967). (14) K. Fuzita, I. Shimokobe, and F. Nakazlma, Nippon Kagaku Kaishi, 8, 1348 (1975). (15) T. H. Gouw and R. E. Jentoft, J. Chromatogr., 68, 303 (1972). (16) T. H. Gouw and R. E. Jentoft, Adv. Chromatogr. 13, 6 (1975). (17) H. K. Newhall, R. E. Jentoft, and P. R. Ballinger, SAE Paper No. 730834 presented at SA€ Meeting, Milwaukee, Wis., September 10-13, 1973. (18) R. E. Jentoft and T. H. Gouw, Anal. Chem., 44, 681 (1972). (19) R . E. Majors, Am. Lab., 4 (9,27 (1972). (20) VYDAC TM Reverse Phase Technical Bulletin 201, The Separations Group, Hesperia, Calif. (21) J. E. Jentoft and T. H. Gouw, J. Chromafogr. Sci., 6, 138 (1970).
(22) (23) (24) (25)
S. T. Sie and G. W. A. Rijnders, Anal. Chim. Acta, 38, 31 (1967). G. W. A. Rijnders., Chem.-lng.-Tech.. 42, 290 (1970). David C. Locke, J. Chromafogr. Sci., 12, 433 (1974). J. Mulik, M. Cooke, M. F. Guyer, G. H. Semeniuk, and E. Sawicki, Prepr, Pap. Natl. Meet., Dlv. Petr. Chem., Am. Chem. SOC., 20, No. 4, 786 (1975).
RECEIVEDfor review March 24,1976. Accepted July 22,1976. Presented in part a t the 170th National Meeting, American Chemical society, Division of Ill. August 26, 1975.
chemistry,
Determination of Homogentisic Acid in Serum and Urine by Liquid Chromatography with Amperometric Detection Paul H. Zoutendam, Craig S. Bruntlett, and Peter T. Kissinger" Department of Chemistry, Purdue University, West Lafayette, Ind. 47907
A new approach is described for the trace analysis of easily oxidized phenolic compounds in biological media. High performance liquid chromatography, uslng a pellicular anion exchange resin and a thin-layer amperometric detector, forms the basis of an improved procedure for 2,5-dihydroxyphenylacelic acid (homogentisic acid, HGA) In blood serum and urine. The very low oxidation potential of HGA permits seleclive and sensitive detection with minimum sample preparation. For serum a detection limit of 1 ng/ml was obtained and a linear response achieved to 100 pg/ml. The overall relatlve standard deviation was found to be 5.6%. A combined TLC-HPLC method for urine was developed for the range 1 pg/ml-100 pg/ml.
The biochemical pathway leading t o t h e catecholamines and their metabolites has received a great deal of attention lately although it represents very minor route in terms of total tyrosine disposition. The major pathway involves production of p -hydroxyphenylpyruvic acid and then 2,5-dihydroxyphenylacetic acid (homogentisic acid or HGA). Under normal circumstances HGA is readily converted by HGA oxidase t o 4-maleylacetoacetic acid and t h e levels of HGA encountered in blood and urine are extremely small. In a few individuals (ca. 1 out of 250 000) the oxidase is not available because of a metabolic defect known as alkaptonuria. This results in dramatic elevation of HGA levels in blood and urine. Alkaptonuria is one of the classical inborn errors of tyrosine metabolism known to every first-year biochemistry student (1). The origin and clinical aspects of the disease have been thoroughly reviewed by Woolf ( 2 ) .The most important analytical studies are those by Neuberger ( 3 ) and by Seegmiller e t al. ( 4 ) .Recent work based on thin-layer chromatography has been reported (5, 6) and a 2,4-dinitrophenylhydrazine colorimetric method has also been described (7). In spite of the low incidence of alkaptonuria, there is interest in following the course of the disease and a highly specific and accurate assay procedure is desirable. Many methods for HGA are based on t h e fact that p - h y droquinones are good reducing agents. Such methods are subject to error due to the presence of other easily oxidizable substances in body fluids. While the enzymatic ( 4 ) ,TLC (5, 6), and some colorimetric (7) procedures partly overcome this 2200
difficulty, they tend t o be insufficiently sensitive to accurately determine serum levels. In the present report we describe a highly selective method based on high performance anion exchange chromatography with amperomeric detection. A potential is chosen which is insufficient to oxidize most other body fluid constituents and therefore the new assay is immune t o many possible interferences. This general approach t o t h e trace analysis of phenols and aromatic amines has been described in previous publications (8-1 0).
EXPERIMENTAL Reagents. All solutions were prepared from reagent grade chemicals in triply distilled water and stored under refrigeration in amber bottles. Aqueous standard solutions of HGA (Sigma Chemical Co.) were prepared by dissolving 10.0 mg HGA in 10.0 ml of 0.1 M acetic acid to give a concentration of I pglpl. Other standards were prepared via appropriate dilutions with the mobile phase (0.5Mor 0.75 M, pH 4.0 acetate buffer). Standard serum and urine samples were prepared by adding relatively small volumes (5-150 pl) of the aqueous standard to between 1.5 and 3.0 ml of serum or urine. The serum and urine were obtained from pooled samples. Apparatus. A previously described liquid chromatography system (11)was used with an electrochemical detector (12) (Bioanalytical Systems Inc., Model LC-10)."Vydac" bonded phase pellicular strong anion exchange resin (The Separations Group) was dry packed in a 50 cm X 2.1 mm i.d. glass column (Altex Scientific, Model 251-02). A previously described precolumn (13) was used. Samples were injected via a Rheodyne Model 50-20 Teflon rotary valve. The valve was used without the manufacturer's sample loop. The connecting tubes and the internal volume of the rotor were calibrated to give an injection volume of 92 p1. Water was circulated through a jacket (Altex Scientific Model 250-10) surrounding the column t o maintain a temperature of 40 "C. The detector potential was usually set at +0.45 V vs. a AgiAgCl reference electrode. Data for the serum assay were acquired via a real time acquisition system centered around a Data General NOVA 2/10 minicomputer. A description of the data acquisition system and the computer program are available on request. Peak heights for the urine assay were obtained manually. Procedure for HGA in Serum. Since HGA readily undergoes air oxidation, all samples should be kept cold and under nitrogen at all times. The nitrogen should be bubbled through all solutions except the raw serum and urine as this causes excessivefoaming.A disposable Pasteur pipet is adequate as a nitrogen bubbler. Place 1.5 ml of 0.066 M HzS04 in 12-ml round bottom polypropylene centrifuge tubes, place the tubes on ice, and bubble nitrogen through each tube. The next few steps should be carried out sequen-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
tially for each individual sample. Add 0.3 ml 10% NaW04. While vortexing the solution, add 0.50 ml serum using an Eppendorf pipet and continue mixing for approximately 20 s. Proteins will precipitate. Immediately return the sample tube to the ice bath, flow nitrogen over the sample, and cap. When these steps have been completed for all samples, centrifuge at 3500 rpm for 15 min. Decant the acidic supernatant into a 12-ml conical glass centrifuge tube into which approximately 0.25 gram NaZS04 has been placed. Pipet 5 ml of ethyl acetate on top of the protein precipitate and immediately pour this into the tube in which the corresponding supernatant was previously placed. Bubble nitrogen through the mixture for approximately 10 s, cap, and place on ice. While keeping on ice, shake the tubes using a reciprocal shaker for 10 min, centrifuge briefly to separate the layers, and transfer the ethyl acetate layer containing HGA into another 12-ml glass centrifuge tube using a Pasteur pipet. Repeat the extraction using another 5-ml portion of ethyl acetate. Evaporate the combined ethyl acetate fractions to dryness under a stream of nitrogen at ropm temperature. An Organomation Associates Inc. N-Evap Model 11I was used for this purpose. Do not immerse the tubes in a water bath during the evaporation! This operation should be started with the first half of the ethyl acetate extract immediately after the first extraction to minimize contact of the samples with oxygen. Take up the residue in 500 pl of the mobile phase. Vortex vigorously, flow nitrogen over the sample, and cap. Take 100 pl of each sample, dilute t o 10.0 ml with mobile phase, flow nitrogen over this solution, and cap. If the original sample contains a higher concentation of HGA than would lie in the linear response range of the electrochemical detector, this second sample can be used. Serum concentrations of HGA greater than approximately 1 pg/ml will require this 1 : l O O dilution. If the samples are to be immediately chromatographed place them on ice, otherwise freeze at -35 "C. It is best to analyze the samples immediately as some decomposition of the HGA occurs even when frozen. Procedure for HGA in Urine. Low levels of HGA are determined in urine by the combination of solvent extraction, thin-layer chromatography, and LCEC. As was noted above, HGA undergoes rapid air oxidation and samples must be kept cold and under nitrogen at all times. The urine samples are acidified with 6 M HCl to pH 2 and stored at -35 OC prior to analysis. A 2-ml aliquot of urine is added to a 12-ml glass centrifuge tube which contains ca. 0.2 g NaCl. The sample tubes are placed in ice and 4 ml ethyl acetate is added, followed immediately by the bubbling of nitrogen through each sample. When the ethyl acetate has been added to all the samples and nitrogen bubbled through each, all samples are shaken for 10 min in an ice bath on a reciprocal shaker. Centrifuge briefly and transfer the ethyl acetate (upper) layer to another 12-ml centrifuge tube and begin evaporating under a stream of nitrogen. Repeat the ethyl acetate extraction two more times combining the additional fractions with the first. The solvent is evaporated to dryness under the stream of nitrogen at room temperature in an air bath. To each sample residue add 200 p1 ethyl acetate and vortex for approximately 45 s. Using a disposable micropipet, spot 20 hl on three of the channels of a prescored silica gel TLC plate (Quantum Industries, LDQ, 5 X 20 cm). The remaining channel on each plate should be used for the spotting of 5 pl of a 1pg/pl standard HGA/ethyl acetate solution. After all samples have been spotted, the TLC plates are developed for 40-45 min using the upper layer of a 2:3:1 benzeneacetic acid-water mixture. Fresh solvent is prepared before each analysis and allowed to equilibrate 1-2 h in a filter lined tank. The plates are removed from the TLC tank and allowed to dry a few minutes before spraying the channel where the HGA standard was placed with Folin and Ciocalteu's phenol reagent. The 3 sample channels are masked during the spraying and the HGA is identified as a blue spot. Using a template made from a clear plastic sheet, remove a 6 mm X 9 mm area of silica corresponding to the HGA on the sample channels. Scrape the silica onto a glassine weighing paper using a razor-sharp straight-edged spatula and transfer to a 12-ml glass centrifuge tube. Blow nitrogen over the scrapings for approximately 20 s, cap, and freeze (-35 'C) until ready to inject on the LC column. The HGA is eluted from the silica gel scrapings by addition of 1.0 ml of the LC mobile phase to the centrifuge tube containing the scrapings, vortexing approximately 30 s, and centrifuging briefly. Aternate Procedure for HGA in Urine. Acidify urine to pH 2 with 6 M HCl and store at -35 "C until ready for analysis. For samples greater than or equal to 1 mg/ml urine, add 1pl (5-pl Hamilton Syringe, No. 85) to 5 ml LC mobile phase (1:5000 dilution) and inject directly on the LC column. For samples less than 1mg/ml urine, dilute the sample by 1000 (e.g., 1 p1 to 1 ml of LC mobile phase) and inject.
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Figure 1. Assay of homogentisic acid in serum at 100 ng/ml Two samples of
the extract (7 ng HGA) were injected using different electrode potentials (uppertrace: E = t0.75 V, lower trace: E = t 0 . 4 5 V). Mobile phase: 0.5 M pH 4 acetate buffer at 0.2 ml/min
RESULTS Serum, Standard curves for HGA in serum were obtained by plotting the peak height (nA) vs. the amount of HGA added to aliquots of the serum pool. The procedure exhibited a linear response from 10 ng/ml t o 100 pglml. The overall relative standard deviation of the method was 5.6%. Recovery relative to HGA placed directly in t h e 0.066 M H2S04-10% NazS04 protein precipitation reagent was 75%. The detection limit was 1ng/ml. At this level the HGA peak-is riding on the tail of t h e void volume peak, making quantitative work difficult. A t present, the detection limit is determined by the finite resolution of the chromatographic separation and limitations of t h e clean-up procedure, not by detector signal-to-noise considerations. HGA could not be detected in serum samples t o which no HGA was added. We d o not believe that this is conclusive proof that HGA does not exist in normal serum in the low ng/ml range, since available sera were not freshly drawn. The small amount of HGA that might have been present could easily have decomposed even though the serum was stored at -35 "C. In any case we believe that t h e present assay is more sensitive by at least two orders of magnitude than t h e previously described methods. Urine. Using t h e combined TLC-LCEC procedure for urine, the response was linear from 1kg/ml to 100 pglml with the lowest detectable amount being 100 ng/ml. T h e relative standard deviation a t t h e 1pg/ml level was 11%and t h e relative recovery 72%. By t h e alternate dilution method, a linear response was obtained from 20 kg/ml t o 4.8 mg/ml. HGA was not detectable in freshly voided urine from healthy individ-
uals.
DISCUSSION Liquid chromatography coupled with electrochemical detection is very specific for the determination of HGA in serum and urine. High performance anion exchange chromatography
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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quantitation of HGA a t this level (100 ng/ml) impossible. Urine is generally a more complicated sample matrix than serum with respect to small molecule metabolites; however, the concentrations encountered are frequently much greater in urine. For example, HGA in the urine of alkaptonuric patients typically ranges from 0.5-5 mg/ml. At this level, and even tenfold lower, it is possible to quantitate HGA with good accuracy by simple dilution followed directly by LCEC (Figure 2). Uric acid (UA) is the only other detectable component normally present a t high levels (11) and it accounts for the small weakly retained peak seen in both the urine samples and blanks. For lower urinary HGA concentrations, greater selectivity is needed. A straightforward approach is to combine TLC with LCEC. The value of this approach has been noted in several earlier reports (9,14) and the scheme is now being used routinely in our laboratory. A TLC sample preparation for HPLC is useful because it can dramatically shorten the time required to process a large number of samples. This is true because samples are studied serially by HPLC but in parallel by TLC. The TLC step can minimize the time required for each HPLC run by eliminating components difficult to resolve or with very long retention times. This approach can be useful even when very efficient microparticle stationary phases are used. The methods described herein are extensions of previous work reported from this laboratory for the determination of trace organic constituents in biological samples. The assay of HGA presents unique advantages (selectivity) and difficulties (instability) when compared with many of the other phenolic compounds we have investigated.
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ACKNOWLEDGMENT The authors thank Lawrence J. Felice for his expert advice
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on urinary acids and Allen L. Schmidt for designing the electronics.
Figure 2. Assay of homogentisic acid in urine (A) HGA (1.4 mg/ml), sample diluted 1:5000. (B) Urine blank diluted 1:5000. (C) HGA (91 pglml), sample diluted 1:lOOO.(D) Urine blank diluted 1:lOOO. Mobile phase: 0.75 M pH 4 acetate buffer at 0.2 ml/min
by itself is inadequate to resolve the components in an extract for quantitation of HGA a t low levels. The redeeming factor in HGA analysis is its oxidation potential, EP,,= +0.43 (14). There are few acids commonly found in serum or urine which have a low oxidation potential a t a carbon electrode (14). By setting the potential of the electrochemical detector to a relatively low value, many possible interferences are not detected. There are certain other easily oxidizable phenolic acids such as caffeic acid, gentisic acid, and 3,4-dihydroxyphenylacetic acid (DOPAC), [E,,, = +0.39, +0.44, and +0.51, respectively (14)] which could possibly interfere with the analysis. Both caffeic and gentisic acids are completely resolved by the HPLC system ( t , = 46 min and > 60 min, respectively). DOPAC is only partially resolved ( t , = 7 min) and could cause significant interference if present in large enough amounts. Figure 1illustrates the virtues of detection a t low potential. The bottom chromatogram was recorded a t a detector potential of +0.45 V. Increasing the potential to +0.75 V reveals the presence of additional electroactive material making
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LITERATURE CITED (1) B. N. La Du, "Alcaptonuria" in "The Metabolic Basis of Inherited Disease", J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson,Ed., McGraw-HIIi, New York, 1972, p 308. (2) L. I. Woolf, Adv. Clin. Chem., 6 , 97 (1963). (3) A. Neuberger, Biochem. J., 41, 431 (1947). (4) J E. Seegmiller, V. G. Zannoni, L. Laster, B. N. La Du, J. B o / . Chem., 236, 774 (1961). (5) J. M Feldman and J. Bowman, Clin. Chem. ( Winston-Salem,N.C.), 19, 459 I1 973). (6) j.Frohiich, G. E. Price, and D. J. Campbell, Clin. Chem. ( Winston-Salem, N.C.), 19, 770 (1973). (7) . . R. E. Stoner and B. B. Blivaiss. Clin. Chem. ( Winston-Salem,N.C.),. 1I . 833 (1965). ( E ) R. M. Riggin, A. L. Schmidt, and P. T.Kissinger, J. Pharm. Sci., 64, 680 (1975). (9) L. J. Felice, W. P. King, and P. T. Kissinger, J. Agric. FoodChem., 24, 380 (1976). (10) R. E. Shoup and P. T. Kissinger, Biochem. Med., 14, 317 (1975). (11) P. T. Kissinger, L. J. Felice, R. M.Riggin, L. A. Pachia, and D. C. Wenke, Clin. Cbem. ( Winston-Salem, N.C.), 20, 992 (1974). (12) R. E. Shoup and P. T. Kissinger, Chem. lnstrum., (in press). (13) L. A. Pachla and P. T. Kissinger, Anal. Chem., 48, 237 (1976). (14) L. J. Felice and P. T. Kissinger. Anal. Chem., 48, 794 (1976).
RECEIVEDfor review July 9,1976. Accepted August 30,1976. Financial support from the National Institute of General Medical. Sciences, the National Science Foundation, and the Showalter Trust Fund is gratefully acknowledged.
DECEMBER 1976
