Anal. Chem. 1906, 58, 2995-2998
problems associated with sigmoidal waves led to the use of derivatives. By taking the mathematical derivative in the electrochemical domain, the waves can be converted to peaks. This is seen in Figure 4b, where the same data set as in Figure 4a has been derivatized and smoothed. The peaks are easier to distinguish, and the difference in the E l j zvalues is more apparent. The tyrosine peak, which was barely visible in Figure 4a, is made quite obvious by the derivatization. The derivative technique improves the visual resolution in the electrochemical domain. This is particularly advantageous in the analysis of complex mixtures. Figure 5 shows the single-potential chromatogram (+1.3 V) and the derivatized chromatovoltammogram of a sample of brewed tea. The many small peaks that are visible in Figure 5 would be difficult to discern if the underivatized chromatovoltammogram were used.
CONCLUSION The changes reported here deal primarily with improvements in the quality of the data obtained using this system. However, these improvements are not applicable to every sample. For trace analysis and quantitative work, the longer electrode in a fixed potential mode has the advantages of higher signal-to-noise ratios and better coulometric efficiency (13). The use of short electrodes and mathematical derivatives is mainly advantageous in qualitative studies. It is important to note, however, that these different modes are easily interchangeable. Electrodes can be made in a variety of lengths. The derivatization is a software choice that is made after data collection is complete. No changes in the system's hardware were required to carry out these studies. Such versatility is a significant advantage of this detection system. ACKNOWLEDGMENT We sincerely thank Robert L. St. Claire I11 for helpful
2995
discussions and for the fabrication of the open-tubular columns used for this study. Registry No. Ascorbic acid, 50-81-7;DL-epinephrine,329-65-7; tyrosine, 60-18-4; dopamine, 51-61-6; hydroquinone, 123-31-9; catechol, 120-80-9.
LITERATURE CITED (1) Samuelsson, R.; O'Dea, J.; Osteryoung, J. Anal. Chem. 1980, 52, 22 15-22 16. (2) Wang, J.; Ouziel, E.; Yarnitzky, Ch.; Ariel, M. Anal. Chim. Acta 1978, 702,99-112. (3) Scanlon, J. J.; Flaquer, P. A.; Robinson, G. W.; O'Brien, G. E.; Sturrock, P. E. Anal. Chlm. Acta 1984, 158,169-177. (4) Reardon, P. A.; O'Brien, G. E.; Sturrock, P. E. Anal. Chim. Acta 1984, 162,175-187. (5) Thomas, M. 5.; Msimanga, H.; Sturrock, P. E. Anal. Chim. Acta 1985, 174,287-291. (6) Stastny, M.; Volf, R.; Benadikova, H.; Vit, I . J . Chromatogr. Sci. 1983, 21, 18-24. (7) Caudill, W. L.;Ewing, A. G.; Jones, S.; Wightman. R. M. Anal. Chem. 1983, 55, 1877-1881. (8) Last, T. A. Anal. Chim. Acta 1983, 155,287-291. (9) Last, T. A. Anal. Chem. 1983, 55, 1509-1512. (10) Kafil, J. B.; Last, T. A. J . Chromatogr. 1985, 348, 397-405. (11) Barnes, A. C.; Nieman, T. A. Anal. Chem. 1983, 55, 2309-2312. (12) White, J. G.; St. Claire; R. L., 111, Jorgenson, J. W. Anal. Chem. 1988, 58,293-298. (13) St. Claire, R . L., 111; Jorgenson, J. W. J . Chromatogr. Sci. 1985, 23, 186-191. (14) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479-482. (15) Savitzky, A,; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1639.
RECEIVED for review May 15,1986. Accepted August 15, 1986. Support for this work was provided by a grant from the Alfred P. Sloan Foundation and the University Research Council of the University of North Carolina. J.G.W. received support from an American Chemical Society Division of Analytical Chemistry Fellowship sponsored by The Perkin-Elmer Corporation. Portions of this work were presented at the 1986 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ.
Digoxin Homogeneous Enzyme Immunoassay Using High-Performance Liquid Chromatographic Column Switching with Amperometric Detection D. Scott Wright,' H. Brian Halsall,* and William R. Heineman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221
The digoxin homogeneous enzyme immunoassay produces NADH as the detectable component for evaluating the drug concentration. The high sensitivity and precision of the amperometrlc detection scheme lead to a substantial decrease in analysis time compared to the conventional spectrophotometric method. High-performance llquid chromatographic column switching is used as an on-line sample cleanup step prior to amperometric detection of NADH. Two columns (aqueous sire-excluslon and reversed-phase C-18) are used. The wfthln-run relative standard deviation for the assay of six replicate serum-based digoxin standards is 2.3 %. The analysis of 46 human serum samples from patients receiving digoxln therapy gives values that correlate well ( r = 0.942) with values (0.6-4.1 ng/mL) obtained on the same samples by radioimmunoassay. Present address: Warner Lambert, Pharmaceutical Research Division, 2800 P l y m o u t h Rd., A n n Arhor, MI 48105.
0003-2700/86/0358-2995$0 1.50/0
The immunoassay has evolved as a, if not the, major technique for the quantitation of biological analytes in complex matrices. Radioimmunoassays, because of their extremely low detection limits, maintain a conspicuous position in the array of detection schemes devised thus far. Nonisotopic immunoassays with detection limits comparable to those of radioimmunoassay (RIA) generally include enzyme amplification. Efforts to develop homogeneous enzyme immunoassays have been based on the principles of competitive binding and most commonly have spectrophotometric detection schemes. Our focus, and that of others, has been to use electrochemical detection for the immunoassay, with our major effort being in amperometric detection ( 1 , 2 ) .A difficulty with electrochemical work using matrices such as serum is the passivation of the electrode surface by components of the matrix. This can be a particularly difficult obstacle in the development of homogeneous assays. The present effort was directed toward using HPLC and column switching to separate electroactive NADH from the 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
complex matrix. This NADH was produced from NAD+ by glucose-6-phosphate dehydrogenase (GGPDH) as amplifier, coupled to digoxin (DIG). Unlabeled digoxin was the physiological analyte. The digoxin assay involves the competition of digoxin and enzyme-labeled digoxin (DIG-GGPDH) for a limiting amount of antibody (Ab) to digoxin as shown below: DIG
Ab:DIG
+
Ab
DIG
Ab:DIG
GBPDH
GBPDH
I
I
-BPGdL
G6P
NAD+
“inactive“
NADH
free
bound
Enzyme-labeled digoxin distributes between two fractions: a free fraction (DIG-GGPDH), which is enzymatically active, and an antibody-bound fraction (Ab:DIG-GGPDH), in which the enzyme activity is diminished by antibody binding. Consequently, the rate of NADH production is directly related to the digoxin concentration. As the amount of digoxin in a sample increases, the amount of free enzyme-labeled digoxin necessarily increases; the result is a greater rate of NADH production. Although there is no separation of bound, labeled digoxin from free, and might therefore be classifiable as homogeneous, the use of a separation step in the measurement of NADH should probably cause this assay to be termed heterogeneous. Although others have used HPLC column switching for the determination of substances in biological fluids (3-5),the method has so far not been coupled to immunoassay schema. Digoxin, a cardioactive drug for the treatment of chronic heart disease, was chosen as the analyte with which to develop this methodology. The low therapeutic level and narrow range, 09-2.0 ng/mL (6), require an assay with good detection limits and good precision. Consequently, digoxin served as an appropriate analyte with which to substantially improve the detection limits of our homogeneous immunoassay with electrochemical detection. This assay was first reported for the determination of phenytoin over the therapeutic range of 0.5-5.0 pg/mL, which is 3 orders of magnitude higher than the range for digoxin (7).
EXPERIMENTAL SECTION Apparatus. A block diagram of the HPLC column switching system with amperometric detection is shown in Figure 1. Two high-pressure solvent delivery pumps (Milton Roy Laboratory Data Control Models 396-31 and 396-57) were used to provide flow rate stability upon column switching. Both pumps delivered mobile phase (0.1 M phosphate buffer, pH 6.6) from a common reservoir at 1.1mL/min. Each flow line had a standing air column pulse damper, a high-pressure gauge, and a presaturator column (22 cm X 4.1 mm or 5 cm X 4.1 mm, Alltech) dry-packed with Vydac pellicular C-18 (Rainin). Two Rheodyne sample injection valves were used for column switching: Model 7125 (20-pL sample loop) syringe loader and Model 7010. Two principal columns were used for column switching: a precolumn (25 cm X 4.6 mm, Knauer) slurry-packed with 10-pm Lichrosorb DIOL (Merck, Darmstadt) and an analytical column (2 cm X 2.0 mm, Upchurch) slurry-packed with 10-pM RSIL C-18 HL (Alltech). A stopwatch was used to time the manual switching of the injection valves. Amperometric detection used flow amperometric equipment from Bioanalytical Systems, Inc., consisting of an LC-4B amperometric detector, a TL-3 thin-layer amperometric flow cell with
Flgure 1. Block diagram of HPLC column switching system with amperometric detection: PD, pulse damper; PS, presaturator column; L, sample loop; 7125 and 7010, sample injection valves; W, waste; PC,
precolumn; AC, analytlcal column; AMP, amperometric detector: and REC, strip-chart recorder. a 5-mil spacer, and an RC-SA reference compartment. The flow cell contained a carbon paste-paraffin oil working electrode, a silver/silver chloride reference electrode (RE-l), and a stainless-steel auxiliary electrode. The amperometric detector was maintained at 750 mV vs. Ag/AgCl. A pipetter-diluter (Model 1500, Fisher Scientific Division) was used to deliver 50 pL of the enzyme standard solution plus 500 pL of phosphate buffer to the reaction tubes maintained at 34 “C. The pipetter-diluter was also used to sample and dilute the enzyme reaction mixtures prior to injecting them into the HPLC column switching system after vortexing. Reagents. EMIT Digoxin assay kits were purchased from the Syva Co. (EMIT (Enzyme Multiplied Immunoassay Technique) is a registered trademark of the Syva Co., Palo Alto, CA.) The immunoassay reagents and calibrators (lot 6H132-17-MOl)were prepared as prescribed in the kit instruction manual. NADH and goat IgG were purchased from the Sigma Chemical Co., uric acid from MCB, and human serum albumin from Miles Laboratories, Inc. The mobile phase (0.1 M phosphate buffer at pH 6.6) was made from Na2HP04and NaH2P0,. Analyses. The EMIT protocol was followed through the production of NADH. In the normal assay, the rate of NADH production is monitored by the absorbance at 340 nm. For the amperometric work, 50 r L of the NADH containing reaction mixture was diluted 11:l with 0.1 M phosphate buffer, pH 7.4, vortexed, and sampled by HPLC. The NADH zone was heart-cut from the chromatographicprofile resulting from passage through the Knauer precolumn and passed through the C-18 column prior to amperometric detection. Under the conditions used, the NADH residence time on the Knauer was 2 min 47 s, and the Knauer column was switched for 20 s permitting NADH transfer. For comparative radioimmunoassays the ARIA HT (Becton Dickinson Immunodiagnostics)was used. Forty-six human serum samples were assayed for digoxin by both the EMIT electrochemical method and the ARIA HT.
RESULTS AND DISCUSSION NADH Detection. As has been reported previously (7,8), NADH is conveniently detected amperometrically by oxidation a t +750 mV vs. Ag/AgCl. A calibration plot a t this potential was linear over the range 0.23-295 pM with a detection limit of 0.06 pM. The injection of higher concentrations caused electrode fouling by the oxidation products. Incubation times for the enzymatic reaction were chosen so that the concentration of NADH produced was below 10 pM. This kept incubation times short, while providing good precision in the data, and prevented electrode fouling by oxidation products. By use of a 5.3 pM solution of NADH, and the column switching protocol, 11consecutive injections yielded a relative standard deviation of only 0.3%. Removal of Electrochemical Interferences. Human serum is an extremely complex matrix and contains a great many electroactive constituents. A consequence of this is that it is difficult to find an electroinactive “window” where an
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table I. Effect of Repeated Injections of Serum Calibrator Samples on Peak Height of NADH
reversed-phase (2-18" reduction in NADH
-
in c1
A
Lichrosorb-DIOL* reduction
in 0 -4
in NADH peak, 7'o
no. of injections
peak, 7'
no. of injections
1 2
39 52 70
1
18
3 13
36 57
r
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B
nSerum calibrator diluted by 112. bSerumcalibrator diluted by 119. au
Table 11. Chromatographic Retention Times"
component
mol w t
retention time
I& HSA NADH uric acid
150000 69 000 709 168
1 min 49 s 1rnin 56 s 2 min 53 s 3 min 7 s
-
Lichrosorb-DIOL column, flow rate 1.15 mL/min, detection by absorbance at 280 nm. indicator analyte such as NADH can be measured relatively free from interferences. In serum, interference to NADH measurement occurs both electrochemically and by passive adsorption a t the electrode surface. Control experiments revealed that reversed-phase (2-18 stationary phase was unable to prevent electrode passivation by serum macromolecules. As shown in Table I, repeated injections of the serum calibrator, which contains NADH and the typical constituents of serum, cause a progressive decrease in the NADH peak due to protein-fouling of the electrode. By comparison, in our assay for phenytoin (7) a C-18 reversed-phase column sufficed to protect the working electrode from passivation by adsorbed proteins for 80-100 sample injections. In that case, however, samples were diluted 80-fold prior to injection in the HPLC because of the much higher concentration of NADH generated in the phenytoin assay. This dilution step reduced interferences from both adsorbing proteins and oxidizable components in the samples. The substantially lower therapeutic level of digoxin prohibits this dilution step, since a much lower concentration of NADH is generated in the digoxin assay than in the phenytoin assay a t corresponding times. A Lichrosorb-DIOL column, however, offers an efficient procedure for separation of proteins from NADH and other small molecules. A representative chromatogram of model macromolecules (passivators), the major electroactive interferent (uric acid), and NADH is shown in Figure 2. The retention times correlate with molecular size as expected in size exclusion chromatography (Table 11). Column switching at ca. 2.5 rnin removes the serum macromolecular passivators and also those purposefully added during the assay procedure. The heart-cut from the NADH zone contains uric acid, which has an of +430 mV vs. Ag/AgCl and therefore interferes with the NADH determination. This is a major interferent, being present at about 300 KM. Passage of the NADH zone through a C-18 reverse-phase column provided a separation factor of 31, easily resolving the two components as shown in Figure 3. This figure also shows the effect of switching time on peak height for five injections of the mixture. The switching times were variable and are indicated above each set of peaks; the timing interval was held constant at 20 s. The left-hand peak of each switching time set is uric acid; the right-hand peak is NADH. The switching time set of 2 min 42 s / 3 rnin 2 s was chosen for operation in the enzyme immunoassay. This set maximizes the NADH response, mini-
2.5
0.0
5.0
W I N J-ES
Figure 2. Chromatogram for serum calibrator-NADH mixture: Lichrosorb-DIOL column: flow rate 1.15 mL/min; detection by absorbance at 280 nm: (A) HSA, I g G (B) NADH; (C) uric acid.
'I
4 "i
'a
r.7
0
0
0 N 0 \
N c1
,
1
'0
N
I
i I------------I I S MIN
Figure 3. Repethive injections of NADH/uric acid mixture (4.00 X lo-' M/2.00 X
M) with different switching times.
mizes the uric acid response, and maximizes the time distance from the HSA retention time. Other timing intervals (from 10 to 90 s) were evaluated; the 20-s interval produced the least electrode fouling and most precise data. A common additive to immunoassay reagents is sodium azide as preservative. The C-18 column is also effective at removing this. Control experiments using column switching revealed that no reduction in the NADH amperometric peak height resulted after 44 serum injections; mean amperometric peak heights for replicate NADH injections ( n = 5 , 4.00 X lo4 M) made
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
n
Figure 4. Assay chromatogram for six digoxin calibrators (0-7.5 ng/mL). Starred peaks represent enzyme-generated NADH. Enzyme reaction sampled at 2 and 7 min for each calibrator: switching time is 2 min 42 s/3 min 2 s.
before and after the serum injections were not statistically = 2.86 nA, different at the 0.10 level of significance (ibefore Sbefore = 0.04 nA; idkf,, = 2.83 nA, S*, = 0.02 nA). Negligible deterioration in chromatographic performance was observed after 176 serum injections; the number of theoretical plates per meter (with respect to NADH and the analytical column) before and after the serum injections were 38 000 and 34 000, respectively. The resolution of NADH and uric acid before and after the injections were 1.1 and 1.0, respectively. The column switching technique is an effective approach for the amperometric measurement of low levels of NADH in serum samples. Enzyme Immunoassay. The EMIT kit for digoxin produces quantities of NADH easily detectable electrochemically in short periods of time. Representative chromatograms for an immunoassay with digoxin calibrators (0-7.5 ng/mL) are shown in Figure 4. Each calibrator was sampled twice (2 and 7 min) with a 5-min interval to provide a simple rate measurement for NADH production. This eliminates the effects of trace interferents, which may coelute with NADH on the C-18 column. The enzymatic generation of NADH during this time interval is evidenced by the substantial increase in NADH peak height for each peak pair. The NADH peak increases with increasing digoxin concentration as expected from the equilibrium scheme shown above. Linear calibration plots have been produced over the range of 0.55-7.5 ng/mL serum dioxin concentration. A typical least-squares calibration equation was I = 1.35 0.16 In C with a correlation coefficient of 0.993;I represents the change in NADH peak current (nA) over the 5-min interval, and C represents the serum digoxin concentration (ng/mL). The ability of the antibody to inhibit enzyme activity of the enzyme-labeled digoxin is poor, and a substantial blank yield of NADH is obtained. This high blank yield is the major contributor to the detection limit obtained, and therefore a high degree of precision for the whole procedure is needed for a useable assay. The relative standard deviation of six replicates of a 2.2 ng/mL digoxin serum standard was 2.370, which is slightly better than those reported for the spectrophotometric or other modifications of the assay, which range from 2.7% to 7.1% (9-14). Under the conditions used, the amperometric method requires a 5-min incubation time interval, whereas the prescribed spectrophotometric method requires 30 min. This substantial shortening in analysis time enables a sample to be analyzed in less than 12 min. Comparison w i t h Radioimmunoassay of Patients' Samples, Forty-six samples of serum from patients on di-
+
goxin therapy were assayed using both the RIA system and the amperometric assay described here. RIA was chosen for comparison rather than the spectrophotometric method in order to reveal the existence of system bias. A correlation plot of the paired data showed good correlation between the two methods, with the least-squares correlation coefficient being 0.94 (slope 0.99 & 0.05, intercept 0.24 f 0.09; the intercept is not significantly different from zero at the 0.01 level of significance). Earlier work (7) was successful in using amperometric detection of enzyme-generated NADH to quantitate phenytoin. Two major differences exist between that and the work described here: the first is the effective use of a simple column switching procedure to essentially eliminate passivating and electroactive interferents. This is important for automated applications, since it reduces column maintenance considerably and the essential principle can be applied to numerous analytes with simple switching program changes. The second difference is the detection level, which here is 1OOOx lower than for the phenytoin and shows the wide dynamic range of these amperometric methods. We recently reported an enzyme-linked immunosorbent assay for digoxin based on the amperometric detection of phenol generated by the enzyme label alkaline phosphatase (15). The detection level of that assay was 50 pg/mL digoxin, which is substantially lower than the detection limit reported here. However, the assay reported here has a detection limit that is certainly adequate for the therapeutic range for digoxin and has the advantage of a much faster analysis time.
ACKNOWLEDGMENT We thank Amadeo J. Pesce and Charles Volle of the University of Cincinnati College of Medicine for their assistance. Registry No. NADH, 58-68-4; digoxin, 20830-75-5. LITERATURE CITED (1) Heinernan, W. R.; Halsail, H. E. Anal. Chem. 1985, 5 7 , 1321A1331A. (2) Doyle, M. J.; Halsall, H. E.; Heinernan, W. R. Anal. Chem. 1984, 56, 2355-2360. (3) Akpofure, C.; Riley. C. A,; Sinkule, J. A,; Evans, W. E. J . Chromatogr. 1982, 232, 377-383. (4) Roth, W.; Beschke, K.; Jauch, R.; Zimrner, A,; Koss, F. W. J . Chromatogr. 1981, 222, 13-22. (5) Little, C. J.; Tornkins, D.K.; Stahel, 0.; Frei, R. W.; Werkhoven-Goewie, C. E. J . Chromatogr. 1983, 264, 183-196. (6) Koch-Weser, J. New Engl. J . M e d . 1972, 287, 227-231. (7) Eggers, M. H.; Halsali, H. E.; Heineman, W. R. Clin. Chem. (WinstonSalem, N.C.) 1982, 28, 1848-1851. (8) Wehrneyer, K. R.; Doyle, M. J.; Wright, D. S.;Eggers, H. M.; Halsali, H. 6.; Heineman, W. R. J . Liq. Chromatogr. 1983, 6 , 2141-2156. (9) Drost, R . H.; Plornp, Th. A,; Teunissen, A. J.: Maas, A. H. J.; Maes, R. A. A. Clin. Chim. Acta 1977, 79, 557-567. I O ) Rurnley, A. G.; Trope, A,; Rowe, D. J. F.; Hainsworth, I . R . Ann. Clin. Biochem. 1980, 77, 315-318. 11) Linday, L.; Drayer, D. E. Clin. Chem. (Winston-Salem, N . C . ) 1983, 29, 175-177. 12) Brunk. S. D.; Malrnstadt, H. V. Clin. Chem. (Winston-Salem, N . C . ) 1977, 23, 1054-1056. 13) Rosenthal, A. F.; Vargas, M. G.; Klass, C . S. Clin. Chem. (WinstonSalem, N.C.) 1976, 22, 1899-1902. (14) Eriksen, P. E.; Andersen, 0.Clin. Chem. (Winston-Salem, N . C . ) 1978, 25, 169-171. Heineman, W. R. Anal. Chem. 1986, (15) Wehmeyer, K. R.; Halsall, H. 6.; 58, 135-139.
RECEIVED for review March 7,1986. Accepted August, 4,1986. Financial support provided by NSF Grant CHE-8217045 is gratefully acknowledged.
