Determination of Homovanillic Acid in Urine by Liquid Chromatography with Electrochemical Detection Lawrence J. Felice and Peter T. Kissinger' Department of Chemistry, Purdue University, West Lafayette, Ind. 47907

A new assay is described for the quantitative determination of 4-hydroxy-3-methoxyphenylacetic acid (homovaniliic acid) in human urine. The precision of the method is f5.1 YO relative standard deviation and the detection limit is approximately 100 pg. A combination of solvent extraction, thin-layer chromatography, and liquid chromatography with amperometric detection provides a highly specific assay. This general approach is thought to be applicable to a wide variety of drug metabolites and natural products.

The importance of homovanillic acid (4-hydroxy-3methoxyphenylacetic acid), the major metabolite of dopamine (3,4-dihydroxyphenylethylamine),is readily apparent from the large number of publications dealing with this compound. Existing methods for the determination of HVA are not entirely satisfactory. One of the earliest approaches and still a widely used method involves the oneelectron oxidation of HVA and measurement of the resulting fluorescent dimer ( I , 2 ) . Besides the usual problems associated with fluorescence measurements, this approach lacks specificity. There are a large number of urinary acids with structures similar to HVA and many of these compounds also form fluorescent dimers upon oxidation (3). More recent assays for HVA employ gas chromatography coupled with electron capture detection or mass spectrometry ( 4 , 5). These methods necessitate the derivatization of HVA to a volatile compound. In addition, the instrumentation required is often not well suited to routine use on large numbers of samples. In our laboratory an assay for urinary HVA has been developed based on the combination of thin-layer chromatography and liquid chromatography with electrochemical detection. The power of thin-layer chromatography to resolve complicated mixtures and its application to a wide variety of biochemical problems is well known. Liquid chromatography with electrochemical detection (LCEC) has been successfully applied to the analysis of many drugs and metabolites in body fluids (6-10). The combination of solvent extraction, TLC, and LCEC results in a highly specific assay procedure for urinary HVA.

EXPERIMENTAL Reagents: (1) hydrochloric acid, 6 M; (2) mobile phase, 4 parts 0.025 M acetate buffer, p H 4.7 and 1 part 0.025 M citrate buffer, pH 5.3; (3) Folin and Ciocalteu's phenol reagent (Sigma Chemical Co.); (4) creatinine standard solution, 1 mg/ml in distilled water; (5) picric acid solution, 1%in distilled water; (6) sodium hydroxide, 10% w/v; (7) standard urine pool. For the standard urine pool, combine urine collected from healthy humans. Acidify to p H 2 with 6 M HC1 and store 20-ml aliquots at -35 OC in glass scintillation vials. Determine the concentration of HVA in the urine pool by the method of standard additions. Analyze the urine pool with and without added HVA. Plot the peak height vs. amount of HVA added and extrapolate to zero peak height to obtain the concentration of HVA in the urine pool. Apparatus. The liquid chromatographic system was similar to that described previously (7) using an electrochemical detector (Bioanalytical Systems, Model LC-2). "Vydac" bonded phase pel794


licular 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). Water was circulated through a jacket (Altex, Model 250-10) surrounding the column to maintain a temperature of 45 "C. The mobile phase was pumped a t a rate of 0.30 ml/min. The detector potential was set at +0.75 V vs. a AglAgCl reference electrode. Cyclic voltammetry experiments were carried out using a Bioanalytical Systems Model CV-1 equipped with a Hewlett-Packard Model 7015 XY recorder. A small (3-mm diameter) carbon paste electrode was used in a simple cylindrical cell with a side arm for a Ad AgCl reference electrode. Details of the electrodes and the cell design will be furnished upon request. Procedure. Acidify urine to pH 2 with 6 M HC1 upon collection and store frozen at -35 "C prior t o analysis. Place 3 ml of urine ( p H 2) in a 12-ml glass centrifuge tube, saturate with NaC1, and add 3 ml of ethyl acetate. Shake for 10 min on a reciprocal shaker, centrifuge briefly, and transfer the ethyl acetate layer to a 6-in. culture tube using a disposable Pasteur pipet. Repeat the extraction with two additional 3-ml volumes of ethyl acetate. Dry the combined ethyl acetate layers over anhydrous sodium sulfate (ca. 1.2 g). Transfer the ethyl acetate to an acid-washed 12-ml centrifuge tube. Wash the residual sodium sulfate with about 1 ml of ethyl acetate and combine with the original extract. Evaporate the solvent to dryness under a stream of nitrogen a t 25 OC. (The use of elevated temperatures at this point results in a significant loss of HVA.) Dissolve the residue in 200 p1 of ethyl acetate and spot 40 pl of this solution onto a prescored silica gel thin-layer plate (Quantum Industries LDQ, 5 X 20 cm), using a 20-pl disposable capillary pipet. Spot the urine extracts on three of the TLC channels and an HVA standard on the fourth channel. Develop the plate with the upper layer of a 100:150:50 benzene, acetic acid, water mixture. Prepare fresh solvent before each assay run using a 250-ml separatory funnel and allow the solvent to equilibrate in a filter paper lined TLC tank 1-2 h in advance. Allow the solvent front to move ca. 15 cm (ca. 45 min), remove the plates, and air dry for ca. 15 min. Mask the channels containing the urine samples with a glass plate and spray the standard HVA channel with Folin and Ciocalteu's phenol reagent. Allow the plate to stand a t room temperature until the HVA is visible as a blue spot (ca. 1h). Using a template made from a clear plastic sheet remove a 12 mm X 10 mm area of silica corresponding to HVA on the sample channels. Scrape the silica onto a glassine weighing paper using a razor-sharp straight-edged spatula and transfer to a conical 12-ml glass centrifuge tube. Add 500 p1 of buffer (LC mobile phase), vortex, and centrifuge. Inject 5 p1 of the supernatant into the LCEC analyzer using a 10-111 Hamilton syringe. Quantitate the HVA by comparing peak heights for the samples to that for the standard urine pool. The level of HVA in casual urine samples is reported as micrograms per milligram of creatinine. Creatinine is determined colorimetrically using the standard picric acid method (11).

RESULTS HVA was determined in 25 urine samples collected from ten apparently healthy males, ranging from 21 to 30 years of age. HVA levels ranged from 0.89 to 8.00 wg/mg of creatinine, which is in general agreement with other investigators (5, 12, 13). Samples were also run after acid hydrolysis of the urine a t pH 1 and 90-95 "C for 15 min. No apparent problems were encountered as a result of acid hydrolysis. Because of the increased levels of HVA after hydrolysis, it is desirable to add 400 ~1 of ethyl acetate to the extract residue, instead of the 200 pl prescribed above.







I. S e l e c t e d P r o p e r t i e s of P h e n o l i c A c i d s t:, min Phenolic acid Rf 0 >60 Chlorogenic 2.8 0.04 3,4,5-Trihydroxybenzoic (gallic) 2.3 0.04 4-Hydroxymandelic 0.05 5.5 2,5-Dihydroxyphenylacetic (homogentisic) 2.2 0.05 4-Hydroxyphenyllactic 0.06 1.8 4-Hydroxy-3-methoxymandelic (vanillomandelic, VMA) 5.0 3,5-Dihydroxybenzoic (a-resorcylic) 0.08 2.5 0.10 3,4-Dihydroxyphenylacetic (DOPAC) 2.8 4-Hydroxy-3-methoxyphenyllactic 0.12 7.0 4-Hydroxy-3-methoxyphenylpyruv- 0.13 icb 0.14 23.1 2,5-Dihydroxybenzoic (gentisic) 0.14 3.2 3,4-Dihydroxybenzoic (protocatechuic) 0.14 1.2 3,4-Dihydroxymandelic (DOMA) 17.0 0.16 3,4-Dihydroxycinnamic (caffeic) 8.0 0.16 5-Hydroxyindoleacetic (HIAA) 4.0 0.16 3,4-Dihydroxyphenylpropionic (dihydrocaffeic) 18.0 0.18 2,3-Dihydroxybenzoic 12.5 0.18 2-Hydroxyhippuric (salicyluric) 0.33 3.5 3-Hydroxyphenylacetic 19.2 0.34 4-Hydroxyphenylacetic 4.0 0.38 4-Hydroxybenzoic 0.39 9.2 2-Hydroxyphenylacetic 6.0 0.39 3-Hydroxybenzoic 17.5 0.41 4-Hydroxycinnamic 6.0 3-Hydroxyphenylpropionic 0.41 5.0 0.41 4-Hydroxyphenylpropionic 3.2 4-Hydroxy-3-methoxyphenylacetic 0.47 (HVA) 7.1 4-Hydroxy-3,5-dimethoxybenzoic 0.54 (syringic) 19.8 4-Hydroxy-3-methoxycinnamic 0.55 (ferulic) 5.3 0.57 4-Hydroxy-3-methoxybenzoic (vanillic) 2-Hydroxybenzoic (salicylic) 0.59 30.0 Table

T 5nA




Figure 1. Typical chromatogram of HVA isolated from human urine by extraction and TLC. A urine pool containing 3.97 Wglml was analyzed in this example

The absolute recovery of HVA was 63 f 5% relative standard deviation and was found to be constant over a concentration range of 1 to 25 kg/ml of urine, The overall precision of the method was found to be f5.1% relative standard deviation. The detector response was measured from 1 to 50 ng injected and the linear calibration in nA vs. ng HVA is described by the following equation:


y = (1.42 f 0 . 0 0 7 ) ~ 0.25 f 0.40(SD)

The detection limit of HVA in an aqueous standard was found t o be 100 pg. A representative chromatogram for a standard urine pool from healthy individuals is shown in Figure 1.

DISCUSSION As can be seen from the Rf values of some common urinary acids (Table I), the TLC step isolates HVA from many possible interferences. TLC has the added advantage that a large number of samples can be processed in parallel and the developing time is only 45 min. Use of the Quantum Industries TLC plates with the preadsorbent spotting area also makes rapid application of the samples possible and improves resolution. Further separation of HVA is then attained by the use of high-performance anion-exchange chromatography. Here the mode of separation is different and the resolution of compounds not adequately resolved by the TLC step is possible. Although the LC mobile phase used gave only marginal resolution, it was chosen because of its relatively rapid elution of HVA (Table I). Greater resolution, a t the expense of time, could be obtained with other mobile phases, such as 0.1 M acetate buffer a t pH 4.7. Finally, the electrochemical detector provides an additional degree of selectivity. Only those compounds which are electroactive a t the carbon paste electrode will be detected. By setting the detector potential a t +0.75 V, further discrimination is obtained. Compounds with oxidation potentials much greater than +0.75 V (Table I) will not give a significant detector response. For example, many of the monophenols listed in Table I have oxidation potentials which are several hundred millivolts greater than f0.75 V and therefore even if they were retained with HVA on the LC column they would not interfere in the assay. Information about one class of compounds such as is given in Table I can be valuable in assessing the possibility of a TLC-LCEC assay for an individual compound. The three factors which dictate the selectivity (i.e., Rf,t,, and EP) are often sufficiently different to ensure an excellent preference for one compound over likely interferences. Manipulation of either one or both mobile phases can, of

+0.38 +0.46 +0.77c +0.43 +OB5 +0.63

+0.83 +0.51 +0.63 +0.23c +0.44 +0.52 +0.56 +0.39 +0.55c +0.42 S0.55

+0.91 C0.88

+OB8 +1.01 f0.86 f0.93 +0.62c +0.88 +0.83 C0.63

+0.56 +0.55



a All samples were ca. 0.5 mM solutions in 1 M acetate buffer at pH 4.7. bPuritydoubtful. Poorly defined anodic peak.

course, increase the probability of success. The present paper describes one of many bioanalytical problems which are amenable to this general approach. Because of the lack of commercial HPLC apparatus specifically designed for the unique requirements of hospital clinical laboratories, it is doubtful that the above procedure will see widespread use in such facilities. At present, the major need for quantitative HVA data is in biomedical research where HPLC is rapidly gaining acceptance. In this context, we believe that the method described here has several important advantages over earlier procedures.

ACKNOWLEDGMENT The authors are grateful to Ralph M. Riggin for his continued interest and helpful suggestions. Craig Bruntlett and William King are thanked for their expert experimental assistance.

LITERATURE CITED (1)D. F. Sharmon. Br. J. Pbarmacol., 20, 204 (1 963). (2) T. L. Sato, J. Lab. Clin. Med., 86, 517 (1965). (3)L. R. Gjessing, E. J. Vellan, B. Werdinius, and H. Corrodi, Acta Chern. Scand.. 21, 820 (1967). (4) E. Anggard, T. Lewander, and B. Sjoquist, Life Sci., 15, 1 1 1 (1974). (5)J. W. Dailey and E. Anggard. Biochern. Pharrnacol., 22, 2591 (1973). ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976


(6) P. T. Kissinger. L. J. Felice, R. M. Riggin, L. A. Pachla, and D. C. Wenke, Clin. Chem. ( Winston-Salem, N.C.).20, 992 (1974). (7) W. D. Slaunwhite, L. A. Pachla, D. C. Wenke. and P. T. Kissinger. Clin. Cbem. ( Wlnston-Salem, N.C.),21, 1427 (1975). (8) R. M. Riggin, A. L. Schmidt, and P. T. Kissinger, J. Pharm. Sci., 64, 680 (1975). (9) L. A. Pachla and P. T. Kissinger, Anal. Cbem., 48, 364 (1976). (10) P. T. Kissinger. R. M. Riggin, R. L. Alcorn. and L.-D. Rau, Biochem. Med., 13, 299 (1975). (11) B. L. Oser, Ed., “Hawk’s Physiological Chemistry”, 14th ed, McGrawHill, New York, N.Y., 1965, p 1040.

(12) G. Ebinger and K. Adriaenssens. Clin. Chim. Acta, 48, 427 (1973). (13) I. Sankoff and T. L. Sourkes. Can. J. Biochem. Physiol., 41, 1381 (1963).

RECEIVEDfor review December 15, 1975. Accepted February 9, 1976. Financial support from the National Science Foundation, The National Institute of General Medical Sciences, and the Showalter Trust Fund is gratefully acknowledged.

High-pressure Liquid Chromatographic Determination of Ascorbic Acid in Selected Foods and Multivitamin Products S. P. Sood,’ L. E. Sartori,* D. P. Wittmer,* and W. G. Haney’ University of Missouri-Kansas City, School of Pharmacy, 5 100 Rockhill Road, Kansas City, Ma. 64 1 10

High-pressure liquid chromatography in the reversedphase, ion-pairing mode has been used to determine ascorbic acid in foods and multivitamin products. While several counterions were investigated for utility, tridecylammonium formate was selected. Workup procedures are minimal, requiring only dissolution of the analyte in water. With detection at 254 nm, solutions of ascorbic acid of 0.5 mg/100 ml can be determined.

The determination of ascorbic acid has been of considerable interest to analysts and was the subject of a recent review ( I ). Classically, 2,6-dichlorophenolindophenolvisual titration ( 2 ) , microfluorometry ( 3 ) ,or colorimetry of the 2,4-dinitrophenylhydrazone of dehydroascorbic acid ( 4 ) have been employed for this determination. However, these methods are often limited by the number of potential interfering substances found in the matrix containing the vitamin (j), and sample workup procedures are therefore complex or results subject to error. In addition, end points are ill defined, and problems with color development and fading are common. Polarographic (6),chromatographic (7), and turbidimetric (8) techniques have also been recently investigated for this determination, but appear also to suffer from the same or related limitations. As a result, a procedure utilizing high-performance liquid chromatography (HPLC) in the reversed-phase mode has been developed and evaluated.

EXPERIMENTAL Apparatus and Operating Conditions. A Model ALC 202 Liquid Chromatograph equipped with a Model 6000 pump and U6K injector (Waters Associates, Milford, Mass.) was used in the study, and column effluents were monitored with the 254-nm detector. Peak areas were determined using an electronic digital integrator (Varian Model 505). The flow rate was 3.0 ml/min. Column. A 30 cm X 4 mm i.d. pBondapak CIS column (Waters Associates) was used. pBondapak Cle has a monomolecular layer of octadecyltrichlorosilane chemically bonded to pPorasil beads having an average particle size of 10 Mm. The number of theoretical plates, based on ascorbic acid at 3.0 ml/min, was 2300, and k o was 1.9 ml. Reagents and Materials. Ascorbic Acid, Reference Standard, was obtained from the U.S.P. Reference Standards Laboratory. Present address, Marion Laboratories, 10236 Bunker Ridge Road, Kansas City, MO 64110. Present address, Waters Associates, Maple Street, Milford, Mass. 01757.




Solutions of the ammonium salts were prepared and used as previously described (9). In brief, a solution of the quaternary ammonium halide in anhydrous methanol was mixed with silver oxide. M) was added Mobile Phase. The ammonium salt (2.0 X to deionized water (300 ml), and the pH was adjusted to 5.0 with 1% formic acid or sodium hydroxide solution. This solution was mixed with an equal volume of methanol and deaerated by vacuum. Calibration Curves. Samples of ascorbic acid of reference grade in phosphate buffer pH 5.0 were prepared to contain 0.5, 1.0, 2.5, 5.0, 7.5, 10.1, and 15.0 mg of ascorbic acid/100 ml. Aliquots (20.0 pl) of these solutions were injected into the chromatographic system, and the resulting peak areas were plotted against concentration for the calibration curve. Sample Preparation. Pharmaceutical Samples. One dosage unit of the sample was homogenized and transferred to a 100 ml volumetric flask with the aid of phosphate buffer pH 5.0 (50 ml). The flask was shaken for 2 min and diluted to volume with the buffer solution. The resulting mixture was filtered, the first 10 ml of filtrate discarded, and an aliquot of the remainder diluted to a final concentration of 5.0 mg/100 ml based on labeled claim. A 20pl. portion of this solution was injected, the resulting peak area determined, and the quantity of ascorbic acid in the sample calculated by reference to the previously derived calibration curves. Foods. A 100-g sample was homogenized with an equal weight of 6% HP03. A portion (10-30 g, accurately weighed) of this slurry was transferred to a 100-ml volumetric flask and diluted to volume with HP03. When necessary, this solution was further diluted to a final concentration range of 0.5-7.5 mg/100 ml. Samples were then treated and ascorbic acid content determined as above.

RESULTS AND DISCUSSION Chmmatographic procedures are a logical choice for the analysis of water-soluble vitamins because of the complex matrices in which they usually occur. Of the quantitative chromatographic techniques, HPLC is more attractive than GLC since derivatives need not be formed prior to analysis, and sample preparation time may therefore be reduced. However, the type of stationary phase employed in HPLC is a crucial factor in maximizing this conceptual advantage. Ion-exchange procedures have been employed in the determination of ascorbic acid ( I O ) , but the ease with which the columns are irreversibly “poisoned” ( 11) necessitates an involved sample preparation scheme. Likewise, normal-phase HPLC (12) suffers from the fact that highly polar constituents of the vitamin sample may be avidly adsorbed to the polar stationary phase. Some of these compounds are eluted only with methanol or water, after which original conditions are difficult, sometimes impossible, to restore. Reverse-phase chromatographic techniques would appear to offer significant advantages in the analysis of

Determination of homovanillic acid in urine by liquid chromatography with electrochemical detection.

Determination of Homovanillic Acid in Urine by Liquid Chromatography with Electrochemical Detection Lawrence J. Felice and Peter T. Kissinger' Departm...
408KB Sizes 0 Downloads 0 Views