with alkyl group size. This decrease, expressed in the values of relative retention volumes, R*,is practically the same for both eluents. The values of equivalent retention volumes, R O , for methylcyclohexane as eluent are lower, which is in accordance with the higher solvent strength in comparison with n-pentane. In Figure 1 are plotted the values of log R o for n-alkylbenzenes and alkylbenzenes symmetrically branched at the a-carbon atom against the number of aliphatic carbon atoms for the two eluents, n-pentane and methylcyclohexane. The R“ values for the rest of the alkylbenzenes which were measured lie between the curve for n-alkylbenzenes and that for alkylbenzenes symmetrically branched at the a-carbon atom. Figure 1 shows very good separation of both types of alkylbenzenes for alkyl chains containing five or more atoms. It is evident that for methylcyclohexane as eluent, both curves approach one another closely, and the separation of alkylbenzenes of different structure becomes more difficult when using a more polar solvent. By means of relations suggested by Snyder ( I , 5 ) for calculation of theoretical retention volumes, Ro for methylcyclohexane, the value eo = 0.01 was used, offering the best results for the series of n-alkylbenzenes. The calculated Rothvalues (see Tables VI and VII) show poorer agreement of experimental and theoretical data for methylcyclohexane than for n-pentane as eluent. However, the results given in Table VI1 confirm conclusions drawn from Table 111,
namely that for alkylbenzenes symmetrically branched at the a-carbon atom, the calculated values are always substantially higher than experimental ones. It might be noted that the method for calculating retention volumes suggested by Snyder ( I , 5) gives very good results in most cases, especially for the system alumina-n-pentane. It seems possible to achieve a good correlation of experimental data by this method, whereas other data such as equivalent retention volumes, R O , or relative retention volumes, R*, depend so much on the sort of alumina and its activity that without the specification of alumina by means of a and V, values, they give only information about the order of eluted solutes. Substantial deviations were observed for alkyl groups symmetrically branched at the a-carbon atom to the ring. In this case the experimental R o values were lower than corresponding R o I hvalues calculated by means of the mentioned method. It seems to be proved that alkyls of this type represent a considerable steric hindrance to adsorption of an aromatic nucleus. Another discrepancy has been found for cycloalkyl groups, where Ro values were practically identical to those for corresponding n-alkyl groups. These results are in contrast to those of Snyder ( I ) , who found substantially higher values. In this case, probably, a different q o j value for a naphthene ring closure is required. RECEIVED for review October 29, 1970. Accepted January 12, 1971.
Radioisotope Derivative Procedure for Determination of Epinephrine or Norepinephrine W. J. Blaedel and T. J. Anderson Department of Chemistry, University of’ Wisconsin, Madison,
Wis.53706
Epinephrine (E) and norepinephrine (NE) are quantitatively and stoichiometrically converted to labelled iodoaminochromes with high specific activity l25I. Inactive iodoaminochrome of the sought-for catecholamine is added, and the activity purified and isolated by column chromatography. E is determinable in the 0.01-1-pM range, and NE in the 0.1-1-pM range, relative precision being 1-2% at midrange. After an alumina adsorption step, the procedure is applicable to the determination of NE in urine. E cannot be determined in urine because of interference by dopamine.
Survey of Methods. There has been much interest for over fifty years in the analysis of epinephrine (E) and/or norepinephrine (NE) (Figure 1) in biological samples ( I ) . Current interest in NE analysis centers around NE’S function as a neurotransmitter in the sympathetic nervous system and its role in brain function and emotional states (2-6). (1) K. Engelman, B. Portnoy, and W. Lovenberg, Amer. J. Med. Sci., 225, 259 (1968). (2) M. Vogt, Brit. J. Pharmacol., 37, 325 (1969). (3) “Second Symposium on Catecholamines,” G. H. Acheson, Ed., The Williams and Wilkins Co., Baltimore, Md., 1966. (4) U. S. von Euler, “Noradrenaline,” Charles C Thomas, Springfield, Ill., 1966. (5) J. R. Crout, Anesthesiology, 29, 661 (1968). (6) J. H. Biel, Aldrichimica Acta, 1, 15 (1968).
Urinary levels of E can be taken as a gauge of the biological activity of the adrenal medulla (5), and greatly increased levels of NE or both compounds indicate a pheochromocytoma, a tumor of the pheochrome system (7). Until recently, the only chemical methods of analysis with sufficient sensitivity and specificity to analyze body fluids for E (0-88nM in urine and about 1 n M in plasma) and for NE (88-310nM in urine and 1-3nM in plasma) have been the ethylenediamine fluorescence method (ED method) and the “trihydroxyindole” fluorescence method (THI method) (8). The ED method is unable to accommodate urine samples because of interference by dopamine (DA), but many THI methods for urinary E or NE exist. Analysis of plasma samples has generally involved using both methods near the limits of their sensitivities ( I , 9, IO), although recent modifications have apparently improved the performance of the THI method (11). Differentiation (7) R. Straus and M. Wurm, Amer. J. Clin. Path., 34, 403 (1960). (8) W. M. Manger, 0. S. Steinsland, G. G. Nahas, K. G. Wakim, and S. Dufton, Clin. Clzem., 15,1101 (1969). (9) S. Udenfriend, “Fluorescence Assay in Biology and Medicine,” Academic Press, New York, N. Y . ,1962 p 149. (10) L. Martin and C. Harrison, A d . Biochem., 25,529 (1968). (11) J. O’Hanlon, Jr., H. C. Campuzano, and S. M. Horvath, Anal. Biocliem., 34, 568 (1970). ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
521
-oApp I
1 EPINEPHRINE
(E)
NOREPINEPHRINE (NE)
DOPAMINE (Dh)
7-IODOADRENOCHROME (ADC-I)
Figure 1. Physiologically significant catecholamines
-o+y-oy+ r
I 7-IODONORADRENOCHROME (NADC-I)
p
7-IODONOREPINOCHROME (NEPC-I)
Figure 2. Iodoaminochromes
between N E and E becomes inaccurate when either compound predominates. Both methods rely on isolation procedures which result in variable and less than quantitative recovery of E and N E from biological samples, both involve frequent standardization, or the establishment of working curves which necessitate strict control of assay conditions, and both are subject to the vagaries of high and variable blanks and fluorescence quenching. The THI method also suffers from fluorescent derivative instability. As a result of the many critical assay variables, some workers have elected to apply continuous methodology to stabilize conditions and procedures in an attempt to attain reproducibility (10, 12-18), Despite the already extensive evolution, modifications of the E D and THI methods continue to appear as a result of the methods’ basic deficiencies (8, I I , 19). A most important development in the analysis of E and N E has been the recent appearance of a number of radiochemical assay methods for these compounds. Saelens et ai. (20) described the first of such assay methods in 1966 based on the enzymatic conversion of N E derived from various brain tissues to epinephrine-N-methyl-14Cusing S-adeno~ylmethionine-rnethyl-~~C and phenylethanolamine-N-methyl transferase. This method, like the E D and THI methods, was dependent on a working curve, was subject to variable loss of N E during isolation, and had the additional disadvantage of being applicable only to samples containing N E at relatively high concentrations (0.1 pg/ml). On the other hand, Engelman er ai. ( I ) developed a doubleisotope derivative assay method which overcame the main objections to the THI and E D methods while introducing some problems of its own. The most significant aspect of this method was the use of tracer amounts of high specific activity NE-3H to determine the overall recovery of “E NE” throughout the procedure and thus to estimate a correction for any loss of E N E which might have occurred. This technique was similar to one previously used by Martin and Harrison (IO) in an automated THI procedure. Engelman et ai. were thus at liberty to employ a number of separation techniques in addition to enzymatic derivatization to attain the requisite selectivity without regard for losses of the derivative. The method was based on the conversion of E and N E to m e t a n e ~ h r i n e - ~and ~ c normetanephrine-I4C
+
+
(12) V. Fiorica, Clin. Chim. Acta, 12, 191 (1965). (13) R. A. Heacock and 0. Hutzinger, Can. J . Chem., 47, 2009 (1969). (14) C. C. Marby and P. H. Warth, Amer. J . Clin. Pathol., 52, 57 (1969). (15) H. McCollough, J . Clin. Pathol., 21,759 (1968). (16) R. J. Merrills, A m i . Biochem., 6, 272 (1963). (17) P. A. Sampson, Jr., U. S. Clearinghouse Fed. Sci. Tech. Inform., 1967. AD 669073. 24 DV. Avail CFSTI. From U. S. Goct. Res. Deuelop. Rep. 68, (ljj, 34 (1968). (18) J. K. Viktora, A. Baukal, and F. W. Wolff, Anal. Biochem., 23, 513 (1968). (19) C. Valori, C. A. Brunori, V. Renzini, and L. Corea. Anal. Biochem., 33, 158 (1970). (20) J. K . Saelens, M. S. Schoen, and G. B. Kovacsics, Biochem. Pharniacol., 16,1043 (1967). 522
CH3
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
by incubation with S-adenosyl-L-methionine-methylF and catechol-0-methyl transferase. The metanephrines were subsequently converted to vanillin- *C and isolated for counting. A later modification of this method (21) allowed independent determination of E and N E and used the tritium tracer of each to determine the recovery of each, overcoming two important deficiencies of the original version. While the method has been successfully applied to both urine and plasma samples, it suffers from a very long and complex procedure, the necessity of quantitating both and 3H in the same solution, the need to determine the absolute specific activity for each batch of S-adenosyl-L-methioninemethyl-’F, and poor accuracy at lower levels of plasma E (20. Two additional, double-isotope derivative methods are those of Aizawa and Yamada (22) and Franklin and Mayer (23, 24). The former used p-toluenesulfonyl chloride-3sS as a derivatizing agent for NE, together with tracer amounts of NE-5H for overall recovery determination in a method for determining NE in rat brain at high levels (0.5 pgjml). Franklin and Mayer described an assay based on the acetylation with a~etic-~H-anhydride of E and NE adsorbed on an alumina column. Isolation by a combination of serial glassfiber paper chromatography and gas chromatography followed. Recovery was determined by using l C-labeled E or NE. The efficacy of the method could not be determined for lack of details and results. All of the above double-isotope derivative methods depend on the use of organic derivatizing reagents which are costly and subject to radiolytic decomposition during storage. Their quantitation of two isotopes in the same solution by liquid scintillation counting necessitates quenching corrections and a set of simultaneous equations for isotope resolution. Also, a compromise is necessary between the amount of 3H or ’ E tracer needed for accurate recovery data and that allowable on the basis of the low endogenous E or N E concentration of the samples. IF or 35S radioactivity is limited to that which allows the accurate determination of 3H radioactivity also present in the sample. Furthermore, the sensitivity of the assays is limited by the relatively low specific activities of the labeled reagents which are available. Outline of the RIDRID Assay. This article describes a promising radioisotope derivative assay method with reverse isotope dilution (RIDRID) for E and NE. It is based on the quantitative conversion of E and N E to their 7-iodo(1261)-aminochromes,iodoadrenochrome (ADC-I) and iodonoradrenochrome (NADC-I), respectively, (Figure Z), by labeled iodine produced in situ by the reaction between peroxymonosulfuric acid (Caro‘s acid) and radioactive KI. Isolation of the radioactive derivative of interest is accomplished by two column chromatography steps, its recovery (21) K . Engelman and B. Portnoy, Circ. Res., 26, 5 3 (1970). (22) Y. Aizawa and K. Yamada, Jap. J . Pharmucol., 19,475 (1969). (23) M. J. Franklin and J. Mayer, Atomlight, 67, 1 (1968). (24) J. S. Stern, M. J. Franklin, and J. Mayer, J . Chromatogr., 30,632 (1967).
being determined by spectophotometrically measuring the recovery of the inactive carrier derivative. To date, the method has precisely and accurately quantitated E (0.01-1 .O b M ) and N E (0.1-1.OpM) in citrate buffer solutions, and its applicability to the determination of urinary N E has been demonstrated. The primary objective has been to develop the measurement procedure, to select working conditions for the assay, and to demonstrate its feasibility for urinary NE assay. DEVELOPMENT AND SELECTION OF ASSAY CONDITIONS
Development and testing of the RIDRID assay method was carried out on simple citrate buffer solutions of E or NE. Under these conditions, the assay can be considered to consist of two parts : the derivative-forming reaction and the separation scheme designed to isolate and measure the radioactive derivative in a pure form. Derivative-Forming Reaction. An iodination reaction is used which is a modification and extension of one first reported by Richter and Blaschko when they isolated ADC-I as a product of the reaction between E and potassium iodate (25). Heacock later established that the product formed under these conditions is 7-iodoaminochrome (26). The kinetics and mechanism of this reaction have been studied (27-30). In the RIDRID assay, iodine formed by the reaction between labeled K I and Caro’s acid reacts with E or NE at pH 5.2 to give quick and quantitative conversion to ADC-I and NADC-I. I n situ production of iodine from a small amount of iodide is an important and unique feature of the method. Were the needed iodine added as iodine, most of it would be reduced to iodide, not only by E and NE, but by even larger amounts of other reducing agents in the biological samples, and very large amounts of lZ6Iactivity would be sacrificed to such reactions. By producing the iodine in situ, any iodide produced by reducing agents in the sample is reoxidized to iodine, again making it available for derivative formation. Caro’s acid has the ability to oxidize iodide to iodine quickly and at low concentrations, apparently maintaining a large concentration ratio of IC to I-, and promoting fast and complete derivatization. Selection of the iodoaminochromes as derivatives is important since it allows the use of 1 2 5 1 as the labeling isotope. 1 2 5 1 decays to ‘ * T e by electron capture, emitting a number of gamma- and X-rays around 30 keV, permitting convenient quantitation in aqueous solution with a gamma-ray spectrometer. Unlike liquid scintillation counting, no quenching corrections are needed. “ 5 1 is available in carrier-free form allowing high sensitivity. Furthermore, the isotope is relatively inexpensive, is chemically stable during storage, and is etTectively shielded by small thicknesses of lead (31). Experimental. REAGENTS.All reagents were analytical grade, and water was triply distilled. Citrate buffers (27mM or 67mM) were prepared by dissolving either 7.93 or 19.7 g of Na,C6H60i.2Hp0in 1 liter (25) D. Richter and H. Blaschko,J . Cliern. Sue., 1937, 601. (26) R. A. Heacock, 0. Hutzinger, B. D. Scott, J. W. Daly, and B. Witkop, J . Anier. Cliein. SOC.,85, 1825 (1963). (27) M. D. Hawley, S. V. Tatawawadi, S. Piekarski, and R. N. Adams, J . Anier. C/ieni.Soc., 89, 447 (1967). (28) R. A. Heacock, Adrair. Hc,trrucyc/. C/7e/n.,5 , 261 (1965). (29) G. L. Mattok and D. L. Wilson, Can. J . Cliem., 45, 1721
(1967). (30) Ibid.,p 2473. (31) P. V. Harper, W. D. Siemens, K . A. Lathrop, and E. Endlich, U.S. A t . Eiiergy Comm.. ACRH-15, 92-103 (1961).
of water, adding 2 ml of methanol (to inhibit microbial growth), and adjusting to pH 5.2 with concentrated HCl. These buffers were stored in a refrigerator. Caro’s acid was prepared fresh daily. About 22.8 mg of fresh (NHa)&08 was dissolved in 1 drop of water. Three drops of concentrated H2S04were added with mixing. One minute later, the mixture was diluted to about 20 ml with water and then 8 drops of 3.2M Na2C03were added with swirling to promote outgassing. The solution was diluted to 50 ml with water, mixed, and in turn, 2 ml of this solution was diluted further to 10 ml with water to give the Caro’s acid reagent (0.4mM). Sodium thiosulfate was 5mM. Potassium iodide stock solution was 0.02M. It was stored in a refrigerator to prevent air oxidation and loss by volatility. Radioactive K I solution was prepared by adding lZ5Ito diluted KI stock solution. First, 25 p1 of K I stock solution was diluted to 10.0 ml with water, giving a 50pM working solution. Two milliliters of this solution were placed in a cylindrical weighing bottle (25 mm X 50 mm) containing a small, glass-sheathed, magnetic stirring bar. About 25 pCi of carrier-free 1 2 5 1 was added in a small volume of solution ( V I ,usually 5 pl) and the solution was stirred to give the radioactive K I solution. The 1 2 5 1 was obtained from Tracerlab, Waltham, Mass., in IO-mCi lots. Upon receipt, it was diluted with about 2 ml of water, so that its activity concentration was around 25 pCi per 5 pl. E and NE sample solutions of known concentration were prepared from the dry solids by weight. E was of USP-grade (Nutritional Biochemicals Corp., Cleveland, Ohio) or USP reference standard E-bitartrate (USP Reference Standards, Bethesda, Md). NE was DL-arterenol.HC1 (NE.HCI), obtained from Sigma Chemical Co., St. Louis, Mo. In every case, the dry solid was accurately weighed, dissolved in pH 5.2, 27mM sodium citrate buffer, and accurately diluted to the desired concentration. The last dilution was done with pH 5.2,67mM citrate buffer in such a way as to prepare about a 3-nil sample of accurately known volume. PROCEDURE. Since the assay calculations involve only ratios of 1 2 5 1 activities, troublesome absolute specific activity measurements were not required. Instead, “count rate specific activities” (a, in counts per minute per milliequivalent) were used, with particular attention paid to the maintenance of constant and reproducible conditions for the measurement of count rates. To estimate the total activity added to the system, the radioactive K I solution was counted. V ml (usually 5 p1) of the radioactive KI solution were delivered into 1 ml of water in a plastic test tube (Cat, No. 2052, Falcon Plastics, Oxnard, Calif.) The Capac micropipetting system (Schwarz BioResearch, Orangeburg, N. Y.)was convenient for this operation. Duplicate counting samples were prepared as a check on the precision of the micropipetting procedure. The count rates of the samples were measured, collecting about 100,000 counts each, with a Model 810C scintillation well-type counter in conjunction with a Model 530 scintillation spectrometer (Baird Atomic, Cambridge, Mass.) and the average count rate (corrected for background) was calculated ( R ) . The count rate specific activity of the iodide (a,) in the radioactive KI solution was then calculated:
where
R
= count rate of radioactive K I solution aliquot
v = VKI
=
counted. volume (ml) of radioactive K I solution aliquot counted-usually 0.005 ml. volume (ml) of diluted (50pM) K I stock solution used-usually 2 ml.
ANALYTICAL CHEMISTRY, VOL. 43, NO, 4 , APRIL 1971
523
VT
volume of carrier-free tracer solution added to prepare the radioactive K1 solution-usually 5 pl. N X I = normality of diluted K I stock solution-usually 50pM. To form the radioderivative, an accurately measured volume (Ve)of the sample whose concentration was to be determined was pipetted into a clean, dry, 25 mm X 50 mm weighing bottle containing a glass-sheathed magnetic stirring bar. Then 1.00 ml of the radioactive K I solution and 1.00 ml of the Caro's acid reagent were added with stirring. About 5 minutes later, 1.00 ml of 5mM Na2S203was added with stirring. Quenching of the derivative-forming reaction with thiosulfate was necessary to reduce all remaining iodine and Caro's acid, to prevent exchange labeling of the inactive carrier added later. Needless delay between thiosulfate addition and carrier addition was avoided: any decomposition and loss of labeled derivative prior to carrier addition is not compensated by the reverse isotope dilution technique. The volume of quenched reaction solution [ V,) was accurately calculable as the sum of the volumes of reagents added. SEPARATION STEP. Following the addition of sodium thiosulfate to the reaction solution, the solution contains the radioactive aminochrome of E or NE, large quantities of radioactive I-, and many unidentified species labeled with IzsI. It is the function of the separation scheme to isolate a measurable fraction of the desired derivative in a radiochemically pure form. Severely low ratios of derivative to nonderivative activities were observed in the assay of simple E or NE solutions, values as low as 1 :lo00 having been encountered. The widely disparate levels of these activities and the need to isolate the lesser component in a radiochemically pure state required a very effective separation method. Biological samples also require the separation of chemically-similar or homolog derivative activities. Such demands led to the development of a double column chromatography separation method for the RIDRID assay. The need for a powerful separation method virtually eliminated the possibility of recovering quantitatively the derivative of interest from the sample. To eliminate the effects of large and variable loss of derivative upon the assay results, a reverse isotope dilution technique was developed which permitted measurement of the derivative recovery in each sample. Excellent precision and accuracy were achieved by sacrificing derivative yield for purity during its isolation. After considerable trial and error, it was found that two strong cation-exchange resin columns (AG5OWX2, 200-400 mesh, Na+-form, BioRad Laboratories, Richmond, Calif.) with water as eluent separated both ADC-I and NADC-I from each other and from nearly all nonderivative activity in buffer samples. The use of a low cross-linkage resin with water as eluent gave fast flush-out of nonderivative activity and minimum spreading of the derivative band. In the two-column process, dilution of the carrier was too great to permit a simple over-all measurement of the carrier yield. Instead, the carrier yield was measured in two steps. A yield was measured after the first column, then more carrier was added, and another yield was measured after the second column. The recovery of the radioactive derivative was determined by measuring the recovery of its chemically equivalent but unlabeled form added to the quenched derivative-forming reaction solution in carrier amounts. The absorbances of the colored ADC-I and NADC-I served for their quantitation. It was not necessary to measure molar absorptivities, since only ratios of carrier concentrations were needed to calculate yields. 524
=
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 197 1
The amount of carrier added to the system at both points in the procedure had to be great enough to permit accurate measurement of carrier absorbances in the effluent of each column, but not great enough to saturate the solution with carrier. Any undissolved carrier would have caused error, by exchange and loss of radioactive derivative on undissolved carrier, and in absorbance measurements. Freeze-drying nearly-saturated but solid-free solutions of either ADC-I or NADC-I proved to be a convenient way of preparing solid carrier in an amount and form which, when reconstituted to a volume slightly larger than its initial volume, resulted in fast dissolution without fear of solution saturation or an insufficient amount of carrier. Since the whole freeze-drying operation was carried out at low temperatures, no problems with derivative stability were encountered. PREPARATION OF FREEZE-DRIED CARRIER.To 160 ml of water, 0.2 gram of E was added and enough concentrated HCl to produce a clear solution. Then, 0.35 gram of KIO, was added, followed by 10 minutes of stirring. One hour after the addition of the KIOI, the crop of crystals was collected by suction filtration through a 0.22-p Millipore filter (Bedford, Mass.), and washed four times with 3- to 4-ml portions of water. The crystals were added to 150 ml of water and stirred vigorously for 1-2 minutes. The solution was suction filtered through another 0.22-p Millipore filter, reserving the filtrate. It was quickly dispensed in 2.2-ml portions from a repeating pipet into 40, lid-less, 10-ml hypodermic syringe vials previously cooled and held at dry ice temperatures. The vials containing the frozen carrier solution were capped loosely with slotted-skirt, rubber hypodermic needle septa and transferred to the cold freeze-drying apparatus. Any convenient freeze-drying apparatus can be used. As soon as the samples were dry, they were stored in a freezer at -20 "C until use. Carrier can be prepared and stored for months in this manner without significant decomposition. The NADC-I carrier was prepared similarly. To 0.1 gram of NE.HC1 in 30 ml of water, 0.16 gram of KIO3 was added, and the solution was stirred for about 10 minutes. One and one-half hours after the KIO3 addition, enough 1M HC1 was added to adjust the pH to 2.0. The solution was then cooled to 4 "C, and after 1/2-1hour, the crystals formed were collected by suction filtration through a 0.22-11 Millipore filter and washed four times with 2- to 3-ml portions of water. The crystals were then added to 150 ml of water, stirred vigorously for 1-2 minutes, and suction filtered through a 0.22-p Millipore filter, reserving the solution. The solution was dispensed in 3.0-ml portions (instead of 2.2-ml portions, to compensate for some mechanical loss of NADC-I during freeze-drying), freeze-dried, and stored in the same manner as ADC-I. PROCEDURE. Immediately after addition of Na2S203 to the reaction solution, about 3 ml of the quenched reaction solution was placed in a vial containing the appropriate freezedried carrier. Simultaneously, a stopwatch was started. A 3-cm square piece of Parafilm (Marathon Corp., Menasha, Wis.) was placed over the mouth of the vial, pressed firmly into place, and held there with the ball of the thumb to obtain a tight seal. Complete dissolution of the carrier was accomplished by a 2-minute agitation with a Vortex Jr. Mixer (Scientific Industries, Queens Village, N. Y . ) . Following dissolution, about 1 ml of the resultant solution was applied to the first cation-exchange column (Lab Crest, Fischer and Porter Co., Warminster, Pa., 5-mm i d . , equipped with Teflon (Du Pont) needle valves, and packed to a depth of 10 cm with 200-400 mesh AG5OWX2 ion-exchange resin
in the Na+ form). Elution was begun with water at 0.2 ml/min. Effluent was collected in 1-ml fractions in plastic test tubes graduated at 1 ml (Falcon No. 2052), and the 1 2 5 1 count rate of each was determined to follow the progress of the elution. The remainder of the quenched reaction solution containing the carrier was placed in a 1-cm absorption cell. The absorbance of ADC-I at 535 nm or the absorbance of NADC-I at 517.5 nm was determined with a Beckman D U Spectrometer with a photomultiplier attachment (Beckman Instrument Co., Fullerton, Calif.). The absorbance of the solution was determined six to eight times with two-minute intervals between observations. The time elapsed between the addition of carrier and each observation was recorded from the stopwatch. This series of absorbance measurements, when plotted as a function of time and extrapolated to carrier addition (zero time), resulted in the initial carrier absorbance (A,) before any significant decomposition had occurred. A linear time dependence permitted easy extrapolation. The carrier decomposed in the quenched reaction solution at a rate sufficient to give a negative error of 1.1-1.5%, had the first absorbance measurement been used in the assay calculations instead of the zero time absorbance. When those three I-ml fractions of the effluent containing the maximum carrier color had been collected, they were combined, mixed well, and the absorbance of the combined effluent (A1) from the first column was determined at the wavelength appropriate to the carrier. The contents of the absorbance cell were emptied into another vial containing freeze-dried carrier and dissolution was accomplished as above. Exactly 1.00 ml of the solution was diluted with 2.00 ml of water, mixed, and the absorbance of the solution (Al ’) at the carrier wavelength was determined. About 1 ml of the carrier-enriched solution was applied to the second cation-exchange column (similar to the first column, but containing a 20-cm resin bed). Elution with water was begun at 0.2 ml/min, effluent again being collected in 1-ml fxactions in graduated plastic test tubes. The lZ5I count rate in each was monitored with a 5-minute counting period immediately after its elution. The values were recorded. Those two 1-ml fractions from the second column containing maximum carrier color were combined, mixed well, and placed in an absorbance cell. The carrier absorbance (AY) of the combined effluent from the second column was read at the carrier wavelength. Exactly 1.00 ml of the solution was placed in a clean plastic test tube and counted until 10,000 counts were collected. The count rate, including background, was calculated and designated as R2. In the case of E analysis, the total nonderivative activity level (R,), including the natural background (Rb) was calculated by averaging the activity contained in milliliters 5-15 of the effluent from the second column (see Discussion Section). In the case of NE analysis, R, was calculated by averaging milliliters 20-25 (see Discussion Section). Thus, in the 1.00 ml of combined effluent from the second column, the count rate due to sample-derived derivative (Rd) was given by :
Assuming quantitative derivatization and identical behavior of the radio and carrier derivatives, the molar concentration ( X ) of E or NE in the original sample solution was calculated: (3)
Figure chromatogram 3. Firstforcolumn an E assay
i,041 I_$
ADC-I Q (L
0 3 ,1 -
0
5
IO
I5
TOTAL EFFLUENT VOLUME, rnl
0 IO 20 30 TOTAL EFFLUENT VOLUME, ml
Figure 4. Second column chromatogram for an E assay Results and Discussion. Figures 3 and 4 are first and second column chromatograms for an E assay at 0.314pM (assay No. 8, Table I). A semi-log plot is used for the first column to show the wide range of activities encountered in the effluent. In both chromatograms, the first peak results from the elution of nonderivative activity while the second is due to elution of the derivative. In Figure 4, the natural background activity Ra is also indicated, demonstrating the excellent derivative to nonderivative activity ratio. Even at the lowest E concentration tested (0.00949pM), the ratio of Rd/(R, - Ra) is 25. Table I presents results of assays of 15 samples of E in citrate buffer solutions from 0.01 to 1pM. Figures 5 and 6 are plots of observed us. known concentrations for each of the two decades analyzed. Least squares analysis of the plots of Figures 5 and 6 show that each is characterized by excellent precision (a relative standard deviation around 1 at midrange). The slopes of both plots are essentially equal, and differ slightly but significantly from unity. Application of these conclusions to the practical analysis of unknown samples will be discussed after results on NE have been presented. ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
0
525
Table I. Assays of E in Buffer Solutions. (0.01-1pM) @I,
9
Observed
Sample E, E, meq R n - Rb, M X lo7 M X lo7 lo-" Rd, cpm cpm 0.122 2.92 50.0 2.0 lb 0.0949 0.327 2.69 127 5.1 2* 0.305 0.535 2.91 258 6.5 3b 0.522 0.779 3.01 358 11 4b 0.777 0.984 5b 0.971 2.59 400 11 1.01 1.79 268 8.0 6 1.02 1.86 1.06 292 5.8 7" 1.94 15 3.04 2.23 975 8 3.14 4.05 9.0 0.855 460 9c 4.37 6.11 1.17 1111 17 10 6.55 1.13 1143 21 1l C 7.65 7.18 8.19 0.650 12 8.51 826 12 8.98 1.22 1524 26 13c 9.35 10.33 1.75 2719 38 14 10.33 11.01 1.25 2140 27 15 11.53 a Sample buffer medium was 67mM citrate. b Samples 1-5 assayed 7 months after samples 6-15, which were assayed over a 5-week period. c Samples were prepared from USP Reference Material E-Bitartrate. All other samples were prepared from Nutritional Biochemicals Corporation's USP grade E. No.
KNOWN E CONCENTRATION,
y (xl0E
Figure 6 . Observed cs. known concentrations for E samples in the range 0.01-O.lpM Least squares analysis: (Samples 1-5 in Table I). Slope, 0.978. Intercept, 0.272 X 10-8M. Std dev, 0.051 X 10-8M
1
121
"I $ IO "
/f
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Figure 5. Observed us. known concentrations for E samples in the range 0.1-1pM
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USP Reference Material E-Bitartrate. Sample No. 7, 9, 11, 13 in Table I 0 USP grade E. All samples in Table I other than 7, 9,11,13 0
Least squares analysis. (Samples 6-15 in Table I. M rejected) Slope, 0.954. Intercept, -0.013 X 10-7M. Std dev, 0.080 X 10-7M. The assays generating Figure 5 were conducted over a period of many weeks while those generating Figure 6 were conducted 7 months after the first set of assays. Excellent time stability of the results is evident. Figures 7 and 8 are the first and second column chromatograms for the assay of a 0.516pM NE sample in citrate 526
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
Sample
Observed !PA NE, NE, meq No. M X lo7 M X lo7 lo-" 1 0.973 0.943 2.05 2 5.16 4.98 3.86 3 9.89 9.50 2.47 a Sample buffer medium was 67mM citrate.
Rd, cpm 784 7658 9925
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buffer (No. 2 in Table 11). They are similar to those for E. Table I1 presents the results of assays of three NE samples in citrate buffer solution in the 0.1-1pM concentration range, and the solid line of Figure 9 is a plot of these data as observed cs. known NE concentrations. As in the case of E assays, the precision was excellent. The slope of the plot was slightly but significantly different from unity, being essentially
the same as the slope of the plots for E. Since the NE content of urine is considerably higher than that of E, the 0.0l-O.lpctM concentration range was not studied. were needed for nonderivative Corrections around 1-2 radioactivities in the regions of the measured peaks (Figures 4 and 8). The correction methods were selected largely on an empirical basis. In the case of ADC-I, the second column pre-peak nonderivative activity levels were about the same as the post-peak levels. So, as a matter of experimental convenience, the nonderivative activity was estimated from the pre-peak region. The pre-peak nonderivative activity not including natural background activity was nearly proportional to the amount of activity applied to the second column in the form of radioactive ADC-I, indicating that decomposition of ADC-I during separation was responsible In the case of NADC-I, the second column pre-peak nonderivative activity levels were much greater than the postpeak levels, and could not be used as a basis for correction. The pre-peak activity levels in the case of NADC-I were closely related to the specific activity of the original K I solution, most likely due to relatively large amounts of impurity activities in the sample applied to the second column. This was apparently due to the fact that the NADC-I peak was eluted considerably sooner than the ADC-I peak. Also, when pre-peak activity levels were used for correction, the least squares line fitting the data of Table I1 was the dotted line of Figure 9, which showed an unacceptably large negative intercept. It should be emphasized that the nonderivative activity level in the peak regions was small relative to the derivative activity, and any error in its estimation had only a small effect on assay results for all except the smallest samples. While the intercepts were of little consequence in the 0.1-1pM regions for both E and NE, the significant difference between the observed slopes (around 0.96) and the theoretical slope of unity had to be dealt with in order to reduce the error of practical assays of real samples. Nurnerous investigations were conducted in an effort to discover the reason for the slope of less than unity. Since both E and NE exhibited identical behavior and since two different sources of E also produced identical results, impurity of the samples, chemical destruction of either the derivatives or the analytes, and incomplete derivatization all seemed unlikely. Rather, a factor common to all assays, regardless of sample concentration or identity, was sought. The spectrophotometer, the gamma-ray spectrometer, and the volumetric glassware were all found to be accurate. Furthermore, an isotopic dilution purity analysis of freeze-dried ADC-I carrier indicated that its recovery from the separation procedure accurately reflected that of the radioactive derivative. The most probable cause appeared to be lack of complete exchange between the lZ6Itracer and the working K I solution to which it was added. Solvent extraction with chloroform on the radioactive KI reagent containing Na2Sz03 and tetraphenylarsonium chloride revealed 3.3 of the iodide to be unextractable. Also, the presence of radioactive particulate matter was demonstrated by filtration through a 0.22-g Millipore filter and radioautography. However, the simple expedient of using filtered radioactive KI reagent for an assay still gave the same results as unfiltered reagent. The high precision and reproducibility of the plots of observed cs. known analyte concentration permits the slope and intercept values obtained from the assay of a few known samples to be used to correct assay results obtained by Equation 3 on unknown samples. Once determined,
Figure 8. Second column chromatoeram for an NE assay
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the correction parameters seem to be independent of working conditions over long periods of time (months) and the batch of lZ5Itracer being used. ANALYSIS OF URINE SAMPLES
Foi application of the RIDRID assay to human urine samples, some additions and changes in reagents and procedure were found necessary. Mainly, a pre-concentration-isolation (PI) step was needed to separate E and NE from the very complex interferences found in urine. Without prior separation, these interferences consumed iodine and Caro’s acid, and prevented formation of ADC-I and NADC-I derivatives. For separation of these interferences, the original work of Shaw (32) has been adopted and modified by most analysts performing urinary E or NE analyses. It is quite specific for (32) F. H. Shaw, Biochem. J.,32,19 (1938). ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
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compounds with a catechol nucleus (33) and numerous PI procedures can be found in the literature. Two serial alumina adsorption steps were needed to reduce the iodine demand of the urine to a point that could be tolerated by the derivative-forming reaction. Incomplete derivatization occurred with only one alumina adsorption step. Other changes in the procedure were made to increase the oxidation and iodination capacity of the derivative-forming reaction, to improve the derivative isolation, and to correct the first extrapolated carrier absorbance value for any inherent absorption of the urine-derived quenched reaction solution at the carrier wavelengths. Apparatus and Reagents. Equipment for the PI step consisted of two Teflon columns, each 5-mm i.d. and 42 mm long, with a porous polyethylene frit press-fitted into the bottom of the column. The top of the column was machined to receive the male end of a 12/1 spherical ground glass joint. Samples were introduced through 50-ml separatory funnels, each equipped with a Teflon stopcock, and terminating with the male portion of a 12/1 ground glass ball joint. To increase the solution flow rates through the column, the top of the separatory funnel was fitted to a nitrogen outlet whose pressure was maintained constant at about 30 cm of water. The alumina adsorbant was Woelm Neutral Activity Grade 1 (Waters Associates, Inc., Framingham, Mass.), processed according to Anton and Sayre (34). Additional solutions required for the PI step were 67mM citric acid, 0.2M EDTA (disodium salt), 0.33M Na2HP0,, 0.5M NaOH, and 5M NaOH. Procedure. URINESAMPLE PREPARATION. Freshly voided urine was acidified with 1 ml of concentrated HCI per 100 g of urine, filtered through Reeve Angel No. 202 filter paper (Clifton, N. J.), and promptly divided into as many 25-ml aliquots as possible. The aliquots were quickly frozen and stored at -20 "C until analysis. Immediately before use, ALUMINA COLUMN PREPARATION. each column was filled with 0.6 gram of processed alumina, tapping to exclude voids. The 0.33M Na2HP04solution was added dropwise to the dry column until it dripped out of the .. (33) "Chromatography," 2nd ed., E. Heftmann, Ed., Reinhold Publishing Corp., New York, N. Y . , 1967, p 71. (34) A. H. Anton and D. F. Sayre, J . Pharmacol. Exptl. Therap., 138,360 (1962). 528
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
bottom. The column was then clamped to the reservoir through the ball joint fittings, using a Parafilm washer for sealing. The stem of the reservoir and the volume above the alumina were filled with 0.33M Na2HP04and the reservoir and column were assembled in such a way as to exclude air bubbles. Then 2-3 ml of 0.33M Na2HP04were allowed to flow through the alumina column, the flow being stopped when the solution meniscus entered the reservoir stem. ADSORPTION.A 25-ml aliquot of the frozen urine sample was brought quickly to room temperature in a water bath. Three milliliters of 0.33M Na2HP04and 1 ml of 0.2M EDTA were added, and the p H was adjusted to 8.4, first using 5M NaOH and then finishing with 0.5M NaOH. The sample was transferred quantitatively to the reservoir, and flow through the column under slight nitrogen pressure was begun at a flow rate around 1 ml/min. When the urine sample meniscus reached the reservoir stem, the stopcock was closed and the reservoir walls were washed with a few milliliters of water. The washings were allowed to pass into the column. Washing was repeated three more times, taking care to exclude air from the column. The column was then unclamped from the reservoir and the solution above the column was allowed to drain completely into the alumina bed. ELUTION. Elution was accomplished with six 0.5-ml portions of 67mM citric acid. Each was allowed to pass completely into the column before the next was added. The column effluent was collected in about 20 ml of 0.06M HC1 containing about 2 mg of ascorbic acid as an anti-oxidant. The diluted effluent from the first column was treated with 3 ml of 0.33M Na2HP04and 1 ml of 0.2M EDTA, the p H was adjusted to 8.4, and it was then subjected to a second alumina adsorption step identical to the first. During elution, the effluent from the second column was collected in a pre-weighed 25 X 50 mm weighing bottle containing a glasssheathed magnetic stirring bar. The pH of the eluate was adjusted to 5.2 with 0.5M NaOH and the volume of the contents was determined by weighing. The pH 5.2 eluate was then passed on to the derivatization step. Since the volumes of the p H 5.2 eluate ( V J and the original urine sample (Vu) were known, the analyte concentration (Xu) in the urine was calculable from Equation 3 , provided that the fraction of the analyte recovered in the PI step (fu) was known: (4) OTHERCHANGES IN THE DERIVATIZATION AND SEPARATION STEPS. Instead of 50pM radioactive KI reagent, lOOpM was used. Also, 2mMinstead of 0.4mM Caro's acid and 10mMinstead of 5mMNa2S203 were used. The urine contributed to the zero time absorbance of the quenched reaction solution. The contribution was found by measuring the absorbance of a portion of the quenched reaction solution to which no carrier was added. The value thus found was subtracted from the zero time absorbance of the quenched reaction solution, determined as described previously. When this correction was omitted, errors around 3 in the value of A , resulted. In the separation step, the length of the first cation exchange column was increased to 180 mm. Results and Discussion. Figure 10 is a second column chromatogram for the assay of NE in urine. (The first column chromatogram is very similar in shape, peak locations, and even peak heights to that in Figure 7 for the assay of NE in citrate buffer solutions.) Also plotted in Figure 10 for each of the fractions isolated in the peak region is the radiopurity (Pt for the jth fraction):
Ri - Rn
p; = ___ A1
Rt is the count rate per milliliter of the fraction, and R, is its nonderivative activity (see preceding text), both including natural background. At is the absorbance of the fraction. It is apparent that Pi is constant for all but the initial stages of the NADC-I elution, indicating that this necessary condition for radiochemical purity of the derivative is met, and that interfering activities are absent. Two recovery experiments were also performed on urine samples-one by adding a k3nown amount of NE after the PI step, and another by adding the NE before the PI step. For the first experiment, three 25-ml portions or urine were processed by the PI step, and the second alumina column eluates were pooled. (Pooling was done to eliminate variations in recovery in the PI step.) To one 3-ml aliquot of the pool, 2.52 nmoles of NE were added (8.39 X lO-TM), while no NE was added to a second 3-ml pool aliquot. Determination of the NE concentration by the RIDRID assay, using Equation 3 and the slope and intercept of Figure 9 as corrections (0.960 and 0.016 X 10-7M, respectively) gave concentrations of 16.76 X 10-7M and 8.38 x lO+M for the NE concentrations in the two aliquots. Thus, of mole per the 8.39 X lop7 mole per liter added, 8.38 X liter (or 99.9 %) was recovered. Another recovery experiment was designed to determine the loss in the PI step. One 25-ml aliquot of a urine sample was analyzed by the PI step and RIDRID assay, and a second 25-ml aliquot was analyzed likewise after adding 5.93 nmoles (2.37 X 10-’M) of NE. Using Equation 4 and the slope and intercept of Figure 9 as corrections, the yield of the PI step (i.e., f u in Equation 4) was calculated to be 65.3%. The native NE concentration, corrected for PI loss, was 2.21 X 10-7M. A repeat on another aliquot of the same urine resulted in 2.07 X lO-’M, indicating that the PI recovery reproducibility was passable, but not comparable to the excellent reproducibility of the RIDRID assay. These values of the native NE concentration compare well with values of 0.88-3.1 X 10-’M reported by others (5). CONCLUSIONS
The RIDRID assay permits the determination of NE in urine with a standard deviation of a few per cent relative. The principal limitation on precision is imposed by the PI step, in which a fairly reproducible loss around 35% is incurred. In practice, the loss is determined by recovery experiments on urine samples, and a correction is then applied to subsequent samples to compensate for the loss. The uncertainty of assuming constancy of loss from sample to sample is obvious. A more satisfactory and precise procedure would be to monitor the loss through the PI step for each sample with a tracer, such as high specific activity tritium-labeled NE. Were the tracer added to the urine immediately, it would not only compensate for PI losses, but also for decomposition during sample storage. Such monitoring has been worked on (1, 10, 21), and should be quite compatible with the RIDRID assay.
The RIDRID assay method in its present form cannot determine urinary E since its aminochrome, ADC-I, displays the same chromatographic behavior in the separation step as the aminochrome of DA, NEPC-I (see Figure 2). D A is present in urine in such quantities, up to 2 p M (35), that its derivative completely masks the E derivative. However, this should not be regarded as a permanent weakness of the method. In light of the identical performance of the derivative-forming reaction in both buffer and urine derived samples for NE, and in light of the fact that buffer solutions of E undergo quantitative derivatization, it is very likely that E is also quantitatively derivatized in urine-derived samples. Successful application of the RIDRID method of urinary E determination awaits the discovery of a more suitable separation system. Development of another separation system also offers two other potential rewards. First of all, sensitivity would be increased by a separation technique which recovered more of the derivatives. This is especially apparent when it is realized that only 0.5% of the sample-derived ADC-I and 1.5% of the sample-derived NADC-I are recovered by the present system. Second, the rather long analysis time could be shortened through the use of a faster separation technique. Modification of the assay for different separation systems should not be difficult, since the derivatization and recovery determination are largely independent of the kind of separation technique used. In comparison with the THI methods of analysis, the RIDRID assay is somewhat slow. However, it is free from the manifold precautions required for low-level fluorescence methods, it is not critically dependent on timing or procedure, it does not require frequent standardization or determination of working curves, and it has the ability to provide a valuable internal check for each sample on its specificity through the use of PI values. The derivative-forming reaction and the reverse isotope dilution technique have worked well in the current method and should provide an excellent base for further development. With improved separation performance, the RIDRID assay method should be able to determine either urinary E or NE and to provide an attractive alternative to either the THI method or the double isotope derivative assay. ACKNOWLEDGMENT
We extend special thanks to John G. Halen for furnishing freeze-drying equipment and for instruction on its use. RECEIVED for review September 3, 1970. Accepted January 4,1971. Work supported by the Atomic Energy Commission under Grant No. AT(11-1)-1082. (35) D. D. Clarke, S. Wilks, S. E. Gitlow, and M. J. Franklin,
J. Gas Chromatogr., 5, 307 (1967).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
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