LITERATURE CITED (1) J. Horowitz, G. D. Gumtz, R. G. Nemchin, and T. P. Meloy, "identification of Oil Spiiis: Comparison of Several Methods", Proc. Joint Conf. Prev. Contr. Oil Spills, 283 (1969). (2) H. Hellmann, Vom Wasser, 41, 45 (1973). (3) L. Melamed, "Oil Source identification Techniques". USNTIS AD Rep 1972, No. 761971. (4) M. E. Garza, Jr., and J. Muth, Environ. Sci. Technol., 8, 249 (1974). (5) I. Lysyj and P. R. Newton, Anal. Chen.~.,44, 2387 (1972). (6) P. F. Lynch and C. W. Brown, Environ. Sci. Technol., 7 , 1123 (1973). (7) W. Coakley, "Comparative Identification of Oil Spills by Fluorescence Spectroscopy Fingerprinting", Proc. Joint Conf. Prev Contr. Oil Spills, 215 (1973). (8) T. Aczel, D. E. Alien, J. H. Harding and E. A. Knipp, Anal. Chem.. 42, 341 (1970). (9) P. M. McElfresh and M. L. Parsons, Anal. Chem., 48, 1021 (1974). (IO) M. S.Vigler and V. F. Gayier, Appl. Spectrosc., 28, 342 (1974). (11) H. R. Lukens, "instruction Manual for Oil Slick identlflcation by Trace Element Patterns Measured with Neutron Activation Analysis", Report 1972, GULF-RT-A- 10973. (12) K. R. Shah, R. H. Filby, and W. A. Haller, J. Radioanal. Chem., 8, 185 (1970). (13) N. A. C. Smith, H. M. Smith, 0. C. Blade, and E. L. Garten, "Bureau of Mines Routine Method for Analysis of Crude Oil. Analytical Method", Bur. Mines. Bull. 490 (1951). (14) D. E. Bryan, V. P. Guinn, R. P. Hackleman, and H. R. Lukens, "Development of Nuclear Analytical Techniques for Oil Slick Identification (Phase I)", USAEC Report GA-9889, Gulf General Atomic Incorporated, Jan. 21, 1970. (15) H. R. Lukens. D. Bryan, N. A. Hiatt, and H. L. Schlesinger, "Develop-
(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26)
ment of Nuclear Analytical Techniques for Oil Slick ldentlfication, Phase IIA", USAEC Report GULF-RT-A-10664, Gulf General Atomic Incorporated, June 11. 1971. E. A. Patrick, "Fundamentals of Pattern Recognition", Prentice-Hall, Inc., Englewood Cliffs, N.J., 1972. H. C. Andrews, "Introduction to the Mathematical Techniques of Pattern Recognltion", Wiley-Interscience, New York, 1972. B. R. Kowaiski, "Pattern Recognition in Chemical Research", in "Computers in Chemical and Biological Research, Vol. 2, C. E. Klopfenstein and C. L. Wilkins, Ed., Academic Press, New York, 1974. B. R. Kowalski and C. F. Bender, J. Am. Chem. SOC.,94, 5632 (1972). B. R. Kowalski and C. F. Bender, J. Am. Chem. SOC.,96, 916 (1974). A. C. Lukens, private communication to T.F.S. B. R. Kowalski and D. L. Duewer, "Documentatlon for ARTHUR (Batch)", Chemometrics Society Report 1, Chemometrics Society, August 1, 1974. N. J. Nilsson. "Learning Machines", McGraw-Hill, New York, 1965. T. M. Cover and P. E. Hart, /E€€ Trans. Inform. Theory, 11-13, 21 (1967). S. Wold, Tech Rep. No. 357, Dept. of Statistics, University of Wisconsin, Madison, Wis. 53706, March 1974. S. Wold, Proc. 2nd Joint Int. Conf. on Pat. Recog., Copenhagen, August 1974.
RECEIVEDfor review February 13,1975. Accepted April 14, 1975. We gratefully acknowledge the financial support of the Office of Naval Research under contract number N00017-67-A-0103-0036, and the National Science Foundation under grant number GP-42013.
Column Radioimmunoassay Method for the Determination of Digitoxin I?.C. Boguslaski and C . L. Schwartz Ames Research Laboratory, Ames Company, Division of Miles Laboratories, he., Elkhart, Ind. 465 14
A rapid, precise, and accurate radioimmunoassay for the measurement of digitoxin In serum samples has been developed. The method employs a column of immoblllzed antlbody which acts as both reaction chamber and separatlon device. A diluted sample is applied to the column and allowed to incubate at room temperature for thirty minutes. Radiolabeled 3- Oauccinyldigltoxigenln tyroslne ( 1251) is then added and incubated for an ldentlcal perlod of time. A buffer wash separates the antlbody bound from free drug. The sensitlvity of the assay is about 150 pg, while the average intra-and Inter-assay coefficients of variation are 10 and 15%, respectlvely. The assay exhibited satisfactory recovery and parallelism and correlated well with a reference procedure; the correiatlon coefficlent was 0.99.
as a guide to drug dosage. Using this rationale, assays have been developed for the measurement of serum or plasma digitoxin (9, I O ) . The range of interest is 5-50 ng/ml. Recently a unique column radioimmunoassay (RIA) procedure for determination of insulin and angiotensin I has been developed (11). The method uses a column of gel entrapped antiserum as a solid phase specific binding reagent. We wish to report the development of a rapid and accurate new method for the quantification of serum or plasma digitoxin which employs a column of covalently bound anti-digitoxin antiserum. The column adds a substantial degree of convenience to the assay since it minimizes transfers by acting as both reaction chamber and separation device.
EXPERIMENTAL Although digitalis has been used for nearly two centuries in the therapy of heart disease, the determination of adequate medication with the drug is still a problem. The extremely narrow therapeutic ratio of this material presents a continuing problem to the clinician. Adverse reactions which were dose-related have been reported to range from 8 to 20% in hospitalized yatients receiving the medication (1-6). Thus, there is need for a method which can be used to monitor patients on digitalis therapy (7). Doherty (8) has shown that there is a rather constant relationship between the plasma and myocardial tissue concentrations of the drug. The plasma concentration, therefore, reflects the myocardial concentration and may serve
Materials.
T h e d i g i t o x i n (reinst) used f o r standards a n d recove r y studies was obtained f r o m Boehringer M a n n h e i m , N e w York, N.Y. Bovine serum a l b u m i n ( B S A ) was obtained f r o m A r m o u r Pharmaceutical, Chicago, Ill.while , C N B r - a c t i v a t e d Sepharose-4B as w e l l as Sepharose-4B was supplied by Pharmacia, Piscataway, N.J. T w e e n 20 was purchased f r o m J. T. Baker, Phillipsburg, N.J., a n d 3-0-succinyldigitoxigenin tyrosine ( l z 5 I ) was obtained f r o m S c h w a r d M a n n , Orangeburg, N.Y. Freund's complete a d j u v a n t was supplied by Difco, Detroit, M i c h . T h e antisera t o d i g i t o x i n was prepared by i m m u n i z a t i o n o f r a b b i t s w i t h a conjugate of t h e steroid glycoside coupled by periodate o x i d a t i o n t o a m i n o groups of lysine in bovine serum albumin. T h e m e t h o d f o r producing t h e conjugate is similar t o published procedures f o r d i g o x i n antisera (12, 13). T h e d i g i t o x i n - B S A conjugate was dissolved in a few drops o f dil u t e sodium hydroxide a n d t h e n P B S b u f f e r was added t o produce ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
1583
1.0 -
m
d
LL:
-0.0 0
z
z m
2
-
0.6
0
~~
Table I. Intra-Assay Precision of Digitoxin Determinations Carried Out on Clinical Specimens
-
Concn range, n g l m l
-
t
k 0.4 V
-
0.2)
I
1
I
5
10
20
I
l
l
x)4050
J
100
DIGITOXIN ng/ml
Flgure 1. Fraction bound as a function to the log of the digitoxin concentration in the sample. The points correspond to the means of six separate standard curves
and t h e bars to one standard deviation
a solution containing 4 mg/ml of conjugate. For immunization, an emulsion was prepared containing equal volumes of the conjugate and complete Freund's adjuvant. Rabbits were initially immunized by injection of 0.1 ml of the emulsion into each of the four footpads and 0.1 ml subcutaneously at each of four sites along the neck and back. This procedure was repeated on the first and third weeks. Following this, beginning on the fifth week and every second week thereafter, the rabbits were injected intramuscularly with 0.4 ml of the emulsion. Beginning with the fourth week, ear bleedings were taken a t monthly intervals. Most of the work presented here was conducted with antiserum obtained from a single rabbit a t the 20th week bleeding. The buffer used throughout this study was phosphate buffered saline (PBS), pH 7.4, prepared from 1.39 g KzHP04, 0.28 g NaH2P04.HzO and 8.77 g NaCl dissolved in glass distilled water to give a total volume of one liter. Before use, Tween 20 was added to the buffer to a concentration of 0.1%. The negative (normal) plasma used in these studies was prepared from outdated blood. The clinical specimens were supplied by the Radioisotopes Department of the South Bend Medical Foundation, South Bend, Ind. They also provided reference assay values following a SchwardMann digitoxin (1251)procedure. Methods. Preparation of the Antiserum-Sepharose Conjugate One gram of CNBr-activated Sepharose-4B was swollen in 200 ml of 0.001N HCl for 15 minutes and filtered under vacuum. The Sepharose was washed with 200 ml of PBS buffer before being resuspended in 4.0 ml of PBS. The Sepharose suspension was carefully stirred and simultaneously treated dropwise with an appropriate dilution of the antiserum in PBS. After the addition was complete, the suspension was rotated for 2 hours a t room temperature and then 22 hours at 4 "C. The Sepharose-antiserum conjugate was washed with 400 ml of PBS. A 50% volume/volume suspension of the conjugate in PBS containing 0.1% soldium azide was then prepared. Preparation of Columns. Support Discs. A cork borer was used to cut discs of approximately 10 mm in diameter and 1.59-mm thickness from a porous sheet of polyethylene (Vyon) obtained from IC1 America, Inc., Wilmington, Del. These discs were washed for approximately 45 minutes in a 1%solution of Brij 35SP. The washed discs were spread and blotted on paper towels before being transferred to a sheet of aluminum foil and dried in an oven a t 90-100 OF for about two hours. Packing of the Column. A treated Vyon disc was inserted into a 3-cm3 Stylex syringe barrel (Pharmaseal Laboratories, Glendale, Calif.) to serve as a support base for the Sepharose. A 50% volume/ volume suspension of the Sepharose-4B-antiserum conjugate was diluted with a 50% suspension of Sepharose-4B to give a final dilution of conjugate which bound approximately 50% of radiolabeled antigen in the absence of unlabeled antigen. Two milliliters of the diluted suspension was added to the syringe barrel and allowed to settle to give a Sepharose column with a bed volume of 1.0 ml. The packed gel was washed with approximately 10 ml of PBS containing 0.1% Tween 20. This buffer was used to prepare all solutions for the assay. 1584
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
0.04.9 5.0-9.9 10.0-19.9 20.0-29.9 3 0.0-3 9.9 40.049.9 50.0-70.5 5.0-49.9
N
14 10 42 43 39 15 16 14 9
h?ean, n g l m l
S.D., ng/mla
1.93 7.64 15.3 24.5 34.9 45.0 56.2 25.5
0.50 0.39 0.78 0.85 0.44 0.66 0.28 0.70
C.V., ohb
25.9 5.1 5.1 3.5 1.3 1.5 0.5 2.7
a S.D. = -where d = difference between duplicates and N = number of pairs. bC.V. = (S.D./Mean digitoxin concn) x
100.
Preparation of Standards. A stock solution of digitoxin in absolute ethanol (5 mg/ml) was prepared and this solution was diluted 1:lOOO with PBS-Tween 20 buffer to give a working solution containing 5 pg/ml. One hundred microliters of this solution was added to 5 ml of normal human plasma to give a solution containing 100 ng/ml. Aliquots of this solution were added to normal human plasma to produce standard solutions containing 0, 5, 10, 20, 30,40, and 50 ng/ml of digitoxin. Digitoxin Assay Procedure. Five-tenths (0.5) milliliter of the appropriate standard or patient's plasma diluted 1 : l O in PBSTween 20 buffer was applied to the column and allowed to incubate for 30 minutes at room temperature. Then 0.5 ml of a 3-0succinyldigitoxigenin tyrosine (lz5I) solution (approximately 10,000 cpm) was applied to the column and allowed to incubate for 30 minutes. During this period the column outlets were capped and an initial count taken in a Gammacord. The Gammacord is about 60% efficient in counting lZ5I. The column was then treated with 10 ml of buffer to separate antibody-bound and free digitoxin. The outlets were again capped and a final count of the column was taken. For ten clinical specimens, the entire procedure including counting can be completed in 90 minutes. Standard curves were constructed by plotting either fraction bound (FB) or logit FB as a function of the log of the concentration of digitoxin in the sample. The following formulas were used to make these calculations.
FB =
cpm in sample o r standard cpm in absence of added digitoxin Logit F B = In-
FB 1 - FB
Values for all clinical specimens were determined from logit standard curves.
RESULTS AND DISCUSSION Precision and Sensitivity of the Standard Curve. The precision of the standard curve was determined from six standard curves run on separate days. All standards were run in duplicate and the mean fraction bound was plotted as a function of the log of the digitoxin concentration in the specimen. Figure 1 shows the mean fraction bound and the inter-assay standard deviation obtained from the separate standard curves. The intra-assay standard deviation was calculated from the difference between the duplicate determinations a t each level. The average intra- and inter-assay coefficients of variation over the range of interest are 10 and 15%,resepectively. During this study, each column was counted individually to obtain an initial count. However, much of this counting is unnecessary and could be eliminated. Data from the inter-assay precision study showed that the coefficient of variation in the percent of label bound for the zero standard was less than 1%,indicating good precision in pipetting of label and preparing columns. The sensitivity of the assay is defined here as the smallest concentration which is greater than, and statistically
CWESTEROL TESrOSTERONE PROOESTERONE CORTISOL ESTRIOL ESTRPDIOL- 178
I
I
l
l
20 30 4050
IO
I
I
100
000
1 lap00
CONCENTRATION (ng/mli
Figure 2.
Cross reactivity of various steroids with the digitoxin antiserum
~~~
Table 11. Parallelism between Specimens a n d Standards Dilution a SerumNo.
None
1 2 3 4
68 78 86 57
a
1:2
68 88 111 76
1:3
1:4
69 81 110 73
62 88 120 80
1:5
1:6
...
83 108 109 71
... 116 78
5 .D ng/Gl
.Mean, n g l m l
C.V.,
7.7 12 12 8.2
70 89 109 72
c
11 13 11 11
The extent to which the specimen was diluted with negative (normal) plasma before being diluted 1 10 with buffer
different from, zero (14). Two standard deviations of the fraction bound at zero concentration correspond to a digitoxin concentration of about 3 ng/ml. Since a 0.5-ml sample diluted 1 : l O is used in the assay, the lowest detectable quantity would be about 150 pg which is more than adequate for the determination of digitoxin concentration in clinical specimens. I n t e r - a n d Intra-Assay Precision Analyses of Clinical Specimens. T o determine the inter-assay precision with clinical samples, five replicate assays were performed on each of four specimens on five separate days. The interassay coefficient of variation was about 15%. The intraassay standard deviation was calculated from the differences between the duplicate results obtained on 179 serum samples. The results given in Table I show that the coefficient of variation over the concentration range 5-50 ng/ml was about 3%. Specificity. The specificity of the antiserum was evaluated by examining the degree of cross reactivity the antiserum exhibited with compounds present in serum or plasma (test compounds) which are structurally similar to digitoxin. A standard curve was generated in the usual manner with digitoxin standards and then standard curves were run for each compound which may cross react. In these studies 3-0-succinyldigitoxigenin tyrosine (lZ5I) was employed as the label, and standards containing various concentrations of the test compounds were used to displace the label from the antibody. The degree of cross reactivity is defined as the concentration of digitoxin needed to give 50% inhibition of the zero value divided by the concentration of test compound needed to give an identical amount of inhibition times one hundred. The result is a percent of cross reactivity for the test compound on a scale where digitoxin is 100%.The concentration of the test compound in serum or plasma determines whether any observed cross reactivity will significantly interfere with the assay ( 1 5 ) . As shown in Figure 2, cholesterol, testosterone, progesterone, estriol, cortisol, and estradiol-17P are unable to displace the labeled digitoxin derivative from the antibody, even when present at concentrations (up to 10,000 ng/ml) well above the normal physiological levels. For purposes of
Table 111. Recovery of Digitoxin Added to Clinical Specimens Amount
Plasma N0.U
1 2 3 4 1 2 3 4 1 2 3 4
Amount
added,
recovered,
nglml
ng/ml
Recovery,
9.92 10.2 9.30 11.4 19.0 19.0 20.4 21.0 38.8 40.6 38.9 40.3
99.2 102. 93.0 114 95.0 95.0 102. 105. 97.0 101. 97.3 101.
10 10 10 10 20 20 20 20 40 40 40 40
Overall average
O1
Aterage,
102
99.2
99.1
100
The endogenous digitoxin concentrations of the specimens were: Plasma 1: 6.2 ng/ml, Plasma 2: 4.05 ng/ml, Plasma 3: 11.0 ng/ml, and Plasma 4: 8.7 ng/ml. a
this assay, these compounds show virtually no cross reactivity with the digitoxin antiserum. However a related cardiac glycoside, digoxin, which is also used in therapy, cross reacts significantly with the antisera. This is not unexpected since the structure of digoxin differs from that of digitoxin only by the presence of a single hydroxyl group. These data indicate that the antiserum cannot discriminate very well between the various cardiac glycosides, implying a need to know which drug has been prescribed prior to assay. Another test of the specificity of a radioimmunoassay is the ability of the method to give the same result at different dilutions of the sample when the actual assay result is multiplied by the dilution factor. This process is termed parallelism since a curve can be generated from the data which is superimposable with the standard curve. Such results suggest that the compound measured in samples from ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
1585
'I
The accuracy of the column RIA method was also tested by analyses of 179 clinical specimens. These samples were assayed previously by a dextran-coated charcoal technique. A comparison of the results from the two assays is given in Figure 3. The correspondence bitween the two sets of data is very satisfactory, the correlation coefficient was 0.99. These results demonstrate that the column RIA procedure is a valid analytical method for the quantification of digitoxin in clinical specimens. It is rapid and possesses acceptable precision, sensitivity, and accuracy. In addition, the method is convenient because both the incubation and separation functions are incorporated into a single device, thus minimizing transfers.
A/' : r = 0.99 SAMPLES
In I
1
I
I
n
I
10
20
30
40
50
60
1
70 DI GI TOXl N (ng/ml)
1
80
DEXTRAN-CQ9TED CHARCOAL PROCEDURE Correspondence of results obtained with the column procedure and the dextran-coated charcoal method Figure 3.
ACKNOWLEDGMENT The authors are grateful to Mrs. Betty Byers of the South Bend Medical Foundation for her kind assistance in sudplying the clinical specimens and reference values. LITERATURE CITED
RIA
patients on digitoxin therapy is immunologically indistinguishable from the digitoxin in the standards. In other words, the antibody recognizes the digitoxin in clinical specimens and standards as identical. As shown in Table 11, acceptable results were obtained in a parallelism study over a sixfold range of dilution. These data indicate that various components in the serum, including potentially cross reacting materials, do not interfere in the test. Accuracy. The accuracy of the assay was evaluated by determining the recovery of exogenous digitoxin added to clinical specimens. Assays were performed on specimens before and after the addition of aliquots of a normal plasma containing known amounts of added digitoxin. The amount of digitoxin recovered from each specimen was then calculated. The results of this analysis given in Table I11 indicate acceptable recovery.
(1)S. Shapiro, D. Slone, G. P. Lewis, and H. Jick, J. Chronic DIs., 22, 361 (1969). (2)I. J. Giuffra and H. L. Tseng, N. Y. State J. Med., 52, 581 (1952). (3)P. L. Rodensky and F. Wasserman, Arch. htern. Med.. 108,171 (1961). (4)A. Schott, Postgrad. Med. J., 40, 628 (1964). (5) M. S.Gotsman and V. Schrire, S.Afr. Med. J., 40,590 (1966). (6)D. W. Duhme, D. J. Greenblatt. and J. Koch-Weser, Ann. Intern. Med., 80,516 (1974). (7)T. W. Smith and E. Haber, Pharmacol. Rev., 25, 219 (1973). (8)J. E. Doherty, W. H. Perkins, and W. J. Flanigan Ann. Intern. Med., 66, 116 (1967). (9)G. C. Oliver, Jr.. B. M. Parker, D. L. Brasfield, and C. W. Parker, J. CIin. Invest., 47, 1035 (1968). (IO) T. W. Smith, J. PharmacoI. Exp. Ther., 175, 352 (1970). (11) S. J. Updike, J. D. Simmons. D. H. Grant, J. A. Magnuson, and T. L. Goodfriend, CIin. Chem., 19, 1339 (1973). (12)T. W. Smith, V. P. Butler. Jr., and E. Haber, Biochem., 9, 331 (1970). (13)V. P. Butler, Jr., and J. P. Chen, Proc. Nat. Acad. Sci. USA, 57, 71 (1967). (14)J. Feldman. and D. Rodbard, "Principles of Competitive Protein-Binding Assays", W. D. Odell and W. H. Daughaday, Ed., Llppincott Co., Philadelphia, Pa., 1971,p 158. (15) C. D. Hawker, Anal. Chem., 45, 878 A (1973).
RECEIVEDfor review February 28,1975. Accepted April 21, 1975.
Determination of Arsenic by Anodic Stripping Voltammetry and Differential Pulse Anodic Stripping Voltammetry Gustaf Forsberg, Jerome W. O'Laughlin, and Robert G. Megargle' Department of Chemistry, University of Missouri, Columbia, Mo. 6520 7
S. R. Koirtyohann Environmental Trace Substances Center, University of Missouri, Columbia, Mo. 6520 7
The determination of arsenlc by anodic stripplng voltammetry (ASV) and differential pulse anodic strlpplng voltammetry (DPASV) was Investigated. Factors affectlng sensitivity and precislon included pH, deposition potential, supporting electrolyte, and the nature of the working electrode. The detection limit of both DPASV and ASV was 0.02 ng/ml. The Present address, Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115. 1586
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
sensitivity increased with Increasing acid concentration and one molar solutlons of either hydrochloric or perchloric acids were most suitable as supporting electrolytes. Gold was found to be superlor to platlnum as a worklng electrode material. The most satisfactory procedure for reduclng arsenlc(V) to arsenic( Ill), whlch Is necessary because arsenic( V) Is electrolnactlve, Involved heating arsenlc( V) wlth NaZS03 In concentrated acid solutions.
