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ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
Determination of Dissolved Iron in Seawater by Radioisotope Dilution and the Chelating Agent Bathophenanthroline G. M. Sharma" and Henry R. DuBois Department of Chemical Oceanography, New York Ocean Science Laboratory, Montauk, New York 11954
A new radioisotope dilution method for the direct determination of ionic iron in a small volume of ocean water is described. Known amounts of unlabeled ferrous ions are added to the allquots of the radioactive ferrous ions ("Fe2+) and the mixtures are reacted with bathophenanthroline to yield [iron-(batho),12+ complex. A standard curve is constructed by plotting % batho-bound radioactivities against the amounts of unlabeled ferrous ions on logit-log paper. The concentration of iron in an unknown sample is obtained by observing the % radioactivity of the labeled iron bound by bathophenanthroline after radiodilution by the unlabeled iron present in a known volume of the sample; the concentration is read from the standard curve. The interference due to cuprous ions is eliminated by complexing them with neocuproine. The method is simple and suitable for routine determination of soluble iron on shipboard.
Although biologically essential trace elements (Fe, Cu, Co, Mn, Zn, Mo, V, etc.) occur a t low concentrations in ocean waters, marine organisms are able to absorb them in quantities far in excess of their biological needs ( I ) . This is well demonstrated by autotrophic algae which contain trace elements a t concentrations as high as lo6 times of their seawater concentrations (2). Biological uptake processes of this magnitude should produce seasonal variations in the distribution of elements being consumed. Experimental verification of this phenomenon would require analysis of a large number of samples per season. Projects of this type would be speeded u p considerably if the analysis could be carried out on shipboard. For this, simple methods for the direct determination of trace elements in a small volume of seawater (1 m L or less) are needed. In this paper we describe a radioisotope dilution method for the determination of ionic iron which enjoys these features. T h e method is based on the observation that after reduction to the ferrous state, the ionic iron in seawater samples or in standard solutions competes with the added radioactive ferrous ions (59Fe2+)for complexing with the iron specific binder, 4,7-diphenyl-l,lO-phenanthroIine (bathophenanthroline or simply batho) on an equal basis. In competitive binding experiments, the radioactivity bound usually represents the degree of isotope dilution (3,4). In view of this we can write Equations 1-3.
B -_
Let BIBo = P, then --P
m
I-P
--
x
P
In ( m )- 2.303 log,, ( x )
EXPERIMENTAL Apparatus. Radioactivity was measured with a Baird Atomic Gamma Counter (Baird-Atomic, Inc., Cambridge, Mass.) equipped with a well type scintillation detector (Model 810), a general purpose scaler (Model 132),and a timer (Model 960A). Reagents used in the assay were pipetted with a Schwarz/Mann "biopipet" or a "dialamatic microdispenser" (calibration 0-100 pL) made by Drummond Scientific Company, Broomall, Pa., (distributor, Fisher Scientific Co.). Logit-log graph papers were purchased from Schwarz/Mann, Mountain View Ave., Orangeburg, N.Y. 10962. Whatman GF/C filter pads for filtering seawater were obtained from VWR Scientific Company. All reactions were carried out in polypropylene tubes. Polypropylene tubes and other plastic apparatus used in assays were cleaned thoroughly by immersing in 50% vjv hydrochloride acid for 2 h and then washing liberally with double-distilled water. Chemicals. Iron-59 solutions containing on the day of calibration -100 pCi of 59Fe as ferric chloride in 1.0 mL of 0.1 N HC1 were purchased from Amersham/Searle Corporation. Total iron in each vial was 13 pg. Bathophenanthroline and neocuproine were purchased from G. Frederick Smith Chemical Company, Columbus, Ohio. Analytical grade sodium acetate, hydroxylamine hydrochloride, and hydrochloric acid were obtained from J. T. Baker Chemical Company. Isoamyl alcohol was purchased from Fisher Scientific Company. Redistilled ethanol was used to prepare bathophenanthroline and neocuproine solutions. Preparation of Reagents. Bathophenanthroline Solution (Batho-solution). A stock batho-solution was prepared by disin 100 mL solving 0.0700 g of 4,'i-diphenyl-l,lO-phenanthroline of ethyl alcohol and then adding 100 mL of distilled water. The stock solution was kept in a well stoppered polyethylene bottle. Working solutions containing 350, 700, and 1230 ng batho-
-
m m f x
&
(substoichiometric amount) of bathophenanthroline is reacted with m units of the labeled ferrous ions and B is t h e radioactivity bound by a units of bathophenanthroline when reacted with a mixture of m units of the labeled and x units of the unlabeled ferrous ions. T h e expressions B / B o = P and m / ( m + x ) represent t h e % batho-bound radioactivity and degree of isotope dilution respectively. When x is known and P is plotted vs. x on a logit-log paper, a linear standard curve is obtained. T h e slope of the line will be -1 when log, x is used or the slope will be -2.303 when log,, x is used. Any large deviation of the slope of t h e experimental line from t h e constant value should indicate interference from some other element or elements to t h e binding of ferrous ions with bathophenanthroline. Using this criterion it was discovered that, except for ionic copper, no other ions present at their normal concentration in seawater compete with ferrous ions for binding to bathophenanthroline. T h e interference due to copper ions was eliminated by complexing them with t h e copper specific binder 2,9-dimethyl-l,lO-phenanthroline (neocuproine). T h e concentration of iron in a n unknown sample is obtained by adding a known volume of the sample to the aliquot of the radioactive iron and observing the percent radioactivity bound by bathophenanthroline from this mixture. T h e concentration is read from the standard curve. T h e sensitivity range of the method appears to extend from the lowest to the highest values of iron encountered in ocean waters. For other applications of this technique, Ref. 5 may be consulted.
(3)
In these equations, Bo is the radioactivity bound when a units 0003-2700/78/0350-0516$01,00/0
C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
phenanthroline in 100 pL of the solvent were prepared by diluting 0.1,0.2, and 0.35-mL aliquots of the stock solution to 10 mL with 50% v/v ethanol-water mixture. These solutions are respectively 2.11 X and 3.68 X M bathophenanthroline. 1.05 X Neocuproine Solution. A stock solution of neocuproine was prepared by dissolving 100 mg of 2,9-dimethyl-l,lO-phenanthroline in 100 mL of ethanol. The working solution was prepared by diluting 1 mL of the stock solution to 10 mL with 50% v/v ethanol-water mixture. Iron-Free Hydroxylamine Hydrochloride. The reagent grade hydroxylamine hydrochloride was crystallized twice from 10 % v/v hydrochloric acid and then from double distilled water. The crystallized material was dried. The dried salt, 10.0 g, was dissolved in 100 mL of distilled water. The solution was made iron-free by reacting with bathophenanthroline and extracting the red iron-batho complex with isoamyl alcohol as described by Strickland and Parsons (6). The iron-free hydroxylamine solution was stored in a well-stoppered polyethylene bottle. Iron-Free Sodium Acetate Buffer. Reagent grade sodium acetate trihydrate, 75 g, was dissolved in 100 mL of distilled water and the solution was made iron-free according to the procedure described by Strickland and Parsons (6). Iron and Copper-Free Seamter. Seawater was filtered through a GF/C filter pad. A 100-mL aliquot of the filtrate was mixed with 10 mL of 0.48 N HC1 and 2.0 mL of hydroxylamine hydrochloride solution in a clean Nalgene separatory funnel and the mixture was allowed to stand for 5 min to complete the reduction of ferric to ferrous and cupric to cuprous ions. Then 2.0 mL of acetate buffer and 5 mL of batho-solution were added and, after mixing, the mixture was let stand for 10 min. The complexes of ferrous and cuprous ions with bathophenanthroline were extracted with 30 mL of isoamyl alcohol. The aqueous layer was separated and boiled in a flask for 10 min to expel most of the isoamyl alcohol. This iron and copper-free seawater was stored in a well-stoppered polyethylene bottle. Iron-59 Solutions. Solutions containing -15 ng, 50 ng, and 100 ng of Fe2+/100pL and having 5000-17 000 cpm/ 100 pL were prepared by mixing appropriate volumes of 10 ppm ferric chloride solution and radioiron solution in 10-mL volumetric flasks. After the addition of 2.0 mL of hydroxylamine hydrochloride solution and 0.4 mL of concentrated HC1, the volumes were brought to 10 mL by adding double-distilled water. The solutions were stored a t 4 “C in well-stoppered plastic tubes. The quantity of hydroxylamine present in these solutions is sufficient to reduce the progressively increasing amounts of ferric ions used in the competitive binding experiments. Cold Iron Solutions. The solutions containing 10, 1, and 0.1 pg ferric ions in 1.0 mL of distilled water were prepared daily from lo00 ppm Fe3+stock solution. The 1000 ppm Fe3+stock solution was prepared by dissolving 0.5000 g of analytical grade iron wire in 20 mL of 6 N HCl and making the volume to 500 mL with distilled water. Copper Solutions. Working solutions containing 10, 1.0, and 0.1 pg of Cu2+in 1.0 mL of distilled water were prepared by serial dilutions of 1000 ppm copper solution purchased from Fisher Scientific Company. Saturation Curve. One milliliter of distilled water was placed in each of the 18 sequentially numbered polypropylene tubes. For duplicate runs, the tubes were arranged in pairs and progressively increasing amounts of radioactive ferrous ions were added to them. After counting the radioactivity in each tube, the pH of the solutions was brought within the range 4-4.5 by adding 50 pL of acetate buffer and mixing. To each tube, 100 p L of 3.68 x M batho-solution was added and, after mixing, the tubes were allowed to stand at ambient temperature for 20 min. During this period the tubes were vortexed three times (after 5 , 10, and 20 min) so that the iron-batho complex produced during the reaction could coat the sides of the tubes. The aqueous solutions were poured into another set of similarly numbered tubes. By measuring the radioactivity in these tubes, the counts due to uncomplexed iron were obtained. Rinsing the assay tubes with water did not improve the precision on counting the uncomplexed iron. The radioactivities bound to bathophenanthroline were calculated by subtracting the counts due to uncomplexed iron from the total counts. The calculated batho-bound radioactivities were almost identical with the ones obtained by counting
517
Table I. Cour1tis.g Data of a Typical Competitive Binding E ~ p e r i m e n t : I r o n=~100 ~ ng; total cpm, 16 7 1 2 Tube No.
0 0 0 10 10 10
1
2 3
4 5 6
7
20 20 20 50
8
9 10 11
50
12 13 14 15
50 100
16
17
200 200
18
200
19 20
500 500
21
500 1000 1000
100 100
22 23 ~
~~~
Cold Fe, ng
Free cpm 5 100 4 948 4 584 6 366 5 906 5 620 6 782 6 392 7 016 8 392 8 242 8 264 10 1 0 2 1 0 080 10 1 3 0 1 2 406 1 2 352 1 2 028 14 728 1 4748 1 4 728 1 6 018 15 890
Bound cpm
BIB,
ll 612
B , = 11835
X
100
l:l 764
12 1 2 8 10 346 10 806 11. 092 9 930 1 0 320 9 696 El 320 8 470 8448 6 610 t3 632 6 582 41 306 4 360 4 684 1984
1964 1984
694 822
87.4 91.3 93.7 83.9
87.2 81.9 70.3 71.6 71.4 55.9 56.0 55.6 36.4 36.8 39.6 16.8 16.6 16.8 5.9 6.9
~~
iron-batho complex directly. The iron-batho complex coated to the sides of the assay tubes was dissolved in 2 mL of isoamyl alcohol prior to counting. The saturation curve shown in Figure 1 was obtained by plotting total counts vs. the bound counts. Competitive Binding Studies. The experiments were done either in duplicate or in triplicate in well-cleaned polypropylene tubes using distilled water, filtered seawater, and seawater made free of iron and copper ions. The amounts of labeled iron used in these experiments are not to be taken too exactly, rather they are intended to indicate a concentration that is orders of magnitude higher than the 1/K value of bathophenanthroline. The concentration of batho-solutions were such that 100-pL aliquot would bind 40-70% of the labeled iron when the dose of the unlabeled iron was zero. Actual amounts of radioactive ferrous ions and bathophenanthroline used in the experiments are listed in the legend of the curves of the experimental data (see Figures 2 and 3). Progressively increasing amounts of the unlabeled iron added to the labeled iron may be read from the abscissa of the graphs. The procedure for obtaining data in triplicate is described below. Water, 1.0 mL, was added to 24 sequentially numbered polypropylene tubes. The tubes were arranged in sets of three, and to sets 2-8 progressively increasing amounts of unlabeled ferric ions were added. Then, 100 pL of iron-59 solution was added to each tube. After mixing, the tubes were allowed to stand at room temperature for 5 min to complete the reduction of the unlabeled ferric ions to the ferrous state by the hydroxylamine present in the iron-59 solution. During this period, the radioactivities in tubes 1-3 were counted and averaged to give total counts for the experiment. Finally, 50 pL of acetate buffer ;and 100 pL of batho-solution of appropriate molarity were added to each tube and the contents of the tubes were mixed thoroughly using a vortex mixer. The procedure for separating the iron-batho complex from the uncomplexed iron was the same as described under “Preparation of the Saturation Curve”. The counts due to uncomplexed iron were determined and subtracted from the total counts to give counts bound by bathophenanthroline. The bound counts in tubes 1 , 2 , and 3 were averaged and the averaged counts were represented by the symbol Bo. The counts bound by bathophenanthroline in all other tubes were represented by the symbol B. Percent radioactivities bound by bathophenanthroline were calculated using the expression P = B/Bo X 100. By plotting P against the amounts of cold iron on logit-log papers, the curves shown in Figures 2 and 3 were obtained. Counting data of a typical experiment and calculation of P from it is shown in Table I.
518
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
Table 11. Inhibition of the Binding of Iron t o Bathophenanthroline b y Copper. Ironi9 = 100 ng; M Solution total cpm = 7751;batho = 100 F L 3.68 X Tube
Cu, ng
Neocuproine, PL
1 2 3
0 0 0
0 0 100
4 5 6 7 8 9
10 10
0 0 100 0 0 100 0 0 100 0 0
17
10 20 20 20 50 50 50 100 100 100 200 200
18
200
10 11
12 13
14 15 16
100 0 0
100
Free cpm
Bound cpm BIB,
3253 3195 3295 3471 3521 3207 4157 3859 3167 4885 4883 3399
4498 4556 4456 4280 4230 4544 3594 3890 4584 2866 2868 4352 1644 1916 4582 294 476 4326
6107
5835 3169 7457 7275 3425
X
100
B o = 4522
94.6 93.5 100.5 79.5 86.1 101.4 63.4 63.4 96.2 36.4 42.4 101.3 6.5 10.5 95.7
Curve B of Figure 3 is the graphical representation of these data. Interference due to Copper. Distilled water. 1.00 mL, and radioiron, 100 ng, were added to each of the 18 sequentially numbered polypropylene tubes. The tubes were arrsnged in sets of three, and to sets 2% known amoimts of cupric ions were added (see Table 11). After mixing, the contents of the tubes were allowed to stand at room temperature for 5 min to complete the reduction of cupric to cuprous ions. During this period the radioactivities in tubes 1-3 were counted and averaged to give total counts used in the experiment. After adding 50 PI>of acetate buffer to all tubes, 100 FL of neocuproine solution was pipetted to one tube of each set (Le,, tubes 3, 6, 9, 12, 15, and 18). This was followed by the addition of 100 FL of 3.68 X 10 M batho-solution to each tube. The experimental procedure for determining the radioactivities bound to bathophenanthroline was the same as described in the preceding experiments. The counting data of this experiment are given in Table 11. The line shown in Figure 4 was obtained by plotting % batho-bound radioactivities vs. the amounts of copper added on a logit-log paper. Preparation of the Standard Curve. The data for the construction of the standard curve was gathered in iron and copper-free seawater. Except for the addition of 100 ILL of neocuproine solution prior to the reaction with bathophenanthroline the procedure for obtaining the data was the same as described under the “Competitive Binding Studies”. The standard curve constructed from the data obtained by reacting 100-pL aliquots of 2.11 X M batho-solution with mixtures of 50 ng of radioactive iron and progressively increasing amounts of the unlabeled iron had a slope of -2.20. Assay Procedure. The seawater samples were filtered through GF/C filter pads to remove particulate iron. One-milliliter aliquots of the filtrates were placed in polypropylene tubes and the same amounts of radioactive iron, acetate buffer, neocuproine and batho-solutions as used in the preparations of the standard curve were added to each tube. The % radioactivities bound to bathophenanthroline were determined and the concentrations were read from the standard curve. These samples were also analyzed by the colorimetric method described in Ref. 6. The results are compared in Table 111. RESULTS AND DISCUSSION T h e determination of iron by competitive binding techniques would require three components: (1)A radioisotope of iron having high specific activity, ( 2 ) a substance that binds specifically and avidly t o ionic iron, and (3) some technique for separating the iron-binder complex from the uncomplexed iron. Preparation of ferric chloride labeled with iron-59, a strong y emitter, are commercially available. T h e bidentate
Table 111. Comparison of the Results of Iron Determination of Seawater by the Radioisotope Dilution Method and by the Colorimetric Method Described b y Strickland and Parsons ( 6 ) Isotopic method (duplicate analysis): PdL 6.0, 4.2 5.0, 4.8 4.1, 4.5 7.0, 7.5 5.2, 5.8
Strickland and Parsons method (single determination),b MiglL
4.9 4.3 5.0 6.0 5.8 a 1.0 mL of seawater was used for each determination. 1 0 0 mL o f seawater was used for each determination by colorimetric method. heterocyclic amine, bathophenanthroline, is known to react with ferrous ions almost specifically to give [Fe(batho),]*+ complex (7). The formation constant ( 8 ) ,K , of the complex is 10’O which suggests that even a t parts per billion level the ferrous ions will react with substoichiometric amounts of bathophenanthroline to give theoretical yield of [Fe(batho)#+ complex. Thus two of the three components needed for t h e radiochemical determination of iron are readily available. In order to develop a technique for separating the ironbatho complex from the uncomplexed iron, known amounts (20-100 ng) of radioactive ferrous ions were reacted with 100-pL aliquots of 3.68 x 10 M batho-solution in polypropylene tubes. Interestingly, t h e iron-batho complex produced during the reaction was found to coat t h e sides of the tubes very tenaciously when the contents of the tubes were swirled using a vortex mixer. T h e aqueous solutions containing the uncomplexed iron were poured into another set of tubes. T h e counts in these tubes were determined and subtracted from t h e total counts t o give counts due to iron-batho complex coated to the sides of the first set of tubes. When batho-bound radioactivities were plotted vs. the total counts, the saturation curve shown in Figure 1 was obtained. This curve clearly demonstrates that even a t low concentrations the reaction between ferrous ions and stoichiometric amount of bathophenanthroline practically goes to completion. The coating of the iron-batho complex to the sides of the assay tubes makes the quantitative separation of the uncomplexed iron from the bound iron a n easy process. Competitions between known amounts of labeled and unlabeled ferrous ions for binding with bathophenanthroline were studied in three separate media: viz, distilled water, seawater, and seawater made free of iron and copper ions. The curves A, B, and C of Figure 2 represent data obtained in distilled water using, respectively, 15, 50, and 100 ng of hot iron. T h e amounts of cold iron added to the aliquots of hot iron may be read from the abscissa of the graphs. T h e slopes of these curves lie in the range -2.20 to -2.30, which is in close agreement with the slope of -2.303 expected for a theoretical curve. In sharp contrast to this the data obtained in filtered seawater gave a curve (Figure 3, curve A) which had a slope of -2.80. T h e increase in the slope of the curve upon going from distilled water to seawater was attributed t o t h e competition of cuprous ions, present in the later medium, with ferrous ions for binding to bathophenanthroline. This view was substantiated when the curve (curve B; Figure 3) constructed from the data obtained in copper-free seawater exhibited a slope of -2.30. I t is well known that neocuproine binds specifically with cuprous ions (7). When the competitive binding experiments were repeated by including this reagent in the protocol, the interference due to copper was found to have been eliminated. T h e data gave curves which had slopes close t o the expected value of -2.303.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
-
Figure 1. Saturation of bathophenanthroline with iron in distilled water. Reactants: batho, 100 FL of 3.68 X ng (1000 cprn 10 ng)
519
M solution; iron59 = 20-100
gg
98
:t
i""
3
I
2
5
20
IO
COLD
50 Fe
100
200
500
1000
(ngl
-
Figure 2. Competitive bindlng curves in distilled water. Curve A: iron5' 15 ng, cprn = 6462; batho, 100 pL of 1.05 X M solution; Bo = 66.5%; slope = -2.30. Curve B: -50 ng, cpm = 11095; batho, 100 pL of 2.11 X M solution; Bc,= 61.8%; slope = -2.20. Curve C: Iron" -100 ng, cpm = 17524; batho, 100 1 L of 3.68 X M solution; B o = 59.4%; slope = -2.20
Unequivocal proof for t h e interference of copper in the binding of iron to bathophenanthroline and for the ability of neocuproine to eliminate this interference was provided by t h e results of the following experiment. Progressively increasing amounts of cupric ions were added to the aliquots of radioiron solution and t h e mixtures were reacted with bathophenanthroline both in the presence and in the absence of neocuproine. T h e counting d a t a of this experiment are collated in Table 111. I t may be noticed t h a t less and less radioactivity binds to bathophenanthroline as the dose of copper added to the radioiron is increased from 0-200 ng. In t h e presence of neocuproine, bathophenanthroline binds the expected amount of radioactivity irrespective of the quantity of copper added to the radioiron. When percent batho-bound radioactivity is plotted vs. added copper on a logit-log paper, the curve shown in Figure 4 is obtained. The linearity of this curve (slope -3.20) suggests t h a t it should be possible to
determine the concentrations of copper in seawater by exploiting the ability of cuprous ions to stoichiometrically reduce the binding of iron to bathophenanthroline. T h e only requirement is that a technique for eliminating the interference due to iron present in seawater should be developed. Work on this problem is being actively pursued. In Figures 2 and 3, 50% dilution of the radioactivity should correspond to a point where the dose of cold iron added is equal to the amount of labeled iron used. I t may be pointed out that in nearly every competitive binding experiment, the quantity of hot iron read from the logit-log plots was - l e 2 5 ng greater than the actual amount of iron-59 used in the experiments. This discrepancy was attributed to the contamination of polypropylene tubes by iron although precautions were taken to clean them thoroughly. Nevertheless, this contamination does not affect the reproducibility of the assay when all the tubes are cleaned in exactly the same way.
520
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978 99
1
I
80
301
-\
2ot 1:
2
5
IO
50
20 COLD
Fe
100
200
500
IdCC
(nu)
Figure 3. Competitive binding curves in filtered seawater and iron and copper-free seawater. Curve A: filtered seawater; iron5' = 16540; batho, 100 pL 3.68 X M solution; B o = 52.5%; slope = -2.80. Curve B: iron and copper-free seawater: iron5' = 16457; batho, 150 pL of 3.68 X M solution; Bo = 71.8%; slope = -2.30
--
100 ng, cpm 100 ng, cpm
98
\
t .\
I
\
I 2
5
20
IO
Cu
50
100
(no)
Figure 4. Inhibition of iron-batho complex formation due to copper. Reactants: iron5' 100 FL of 3.68 X M solution: B o = 58.3%; slope = -3.20
The tubes cleaned in identical fashion are expected to contain the same quantity of residual iron. I n the procedure €or the preparation of the standard curve and determination of iron in unknown samples, neocuproine was used to complex copper ions which otherwise interfere with the binding of iron to bathophenanthroline. Several seawater sampies were analyzed for the concentration of the dissolved iron by the isotopic method and by the colorimetric method described in the literature (6). The results (Table 111) indicated a fair agreement between the two methods. It may be pointed out that, to approximate shipboard conditions, we carried out all experiments in a laboratory which was neither air conditioned nor kept solely for trace element work. T h e major source of contamination, under these conditions, was the air-borne dust which completely voids the assay if the tubes are not kept capped a t all times except for the addition of reagents. With this precaution, the values obtained in replicate determinations agreed well among
200
-
500
1000
100 ng, cprn = 7751; copper = 0-200 ng; batho,
themselves in the range 10-100 ng Fe/mL. The relative standard deviation at 10 and 20 ng Fe/mL was 13.6 and 12.470, respectively. In the range around 1-5 ng Fe/mIJ level, the scatter of individual determinations around the mean was found t o be much larger if all sources of contamination were not scrupulously eliminated. This point is well demonstrated by the data reported in Table III.
LITERATURE CITED ( 1 ) H. J . M. Bowen, "Trace Elements in Biochemistry", Academic Press, London and New York, 1966, pp 68-69. (2) J. P. Riley and R. Chester, "Introduction to Marine Chemistry", Academic Press, London and New York, 1971, p 92. (3) A. Zettner, Clin. Chem. (Winston-Sabrn, N.C.). 19, 699-705 (1973). (4) A. Zettner and P. E. Duly, Clin. Chem. ( Winston-Salem, N.C.),20, 5-14 I.?,-..\
(1514).
( 5 ) J. Ruzicka and J. Stary, "Substiochiometfy in Radiochemical Analysis", Pergamon Press, Oxford, 1968. (6) J. D. H. Strickland and T. R. Parsons. "A Practical Handbook of Seawater Analysis", J. C . Stevenson, Ed., Fisheries Research Board of Canada, Ottawa, 1968, pp 99-107.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978 (7) J. P. Riley
"Chemical Oceanography,"Voi. 2, J. P. Riley and G. Skirrow. Ed., Academic Press, London and New York. 1965, pp 378-381, 384. (8) E. Konig, Coord. Chem. Rev., 3, 471-495 (1968).
RECEIVED for review September 6, 1977. Accepted January
521
3,1978. This work was supported by New York State Contract No. C108738 and by grants from Nassau and Suffolk Counties in New York State. New York Ocean Science Laboratory Contribution No. 83.
Determination of Phentolamine in Blood and Urine by High Performance Liquid Chromatography Frederic de Bros" Anesthesia Laboratory of Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02 1 14
Ernest M. Wolshin' Astra Pharmaceutical Laboratory of Clinical Pharmacology, St. Vincent Hospital, Worcester, Massachusetts
A high performance liquid chromatographic method for the analysis of phentolamine in blood and urine is described. The procedure requires 1 mL of biological fluids and involves the addition of a double internal standard, alkalinization, extraction into ether, followed by back extraction into sulfuric acid. Separation of drug and internal standards is accomplished by HPLC on a reverse phase column with octane sulfonic acid in the mobile phase as an ion pairing reagent. Chromatographfc separation is complete in less than 12 minutes. The assay is linear for concentrations from 15 to 5000 ng/mL. The limit of detection is 15 ng/mL for a 1-mL sample. Relative standard deviations for replicate samples average 4.57 YO,The assay Is specific for phentolamine. There is no interference from commonly coadministered drugs.
is time consuming but may be of value for monitoring of drug concentrations in urine. Although gats-liquid chromatography is employed to assess the purity of pharmaceutical preparations and possibly may be adaptable t o clinical determinations, the technique for derivatization of phentolamine and its measurement in nanogram quantities has not been reported. A rapid and specific, clinically applicable assay with nanogram sensitivity for phentolamine has not previously been published. This paper presents a n analytic method which meets the above criteria for phentolamine in biological fluids. I t can measure the drug in a broad concentration range and is suitable for pharmaceutical development purposes as well as drug monitoring in a clinical laboratory.
EXPERIMENTAL P h e n t o l a m i n e o r 2-(N'-p-tolyl-N'-n-hydroxyphenylaminomethy1)imidazoline (Figure 1) is a n LY adrenergic blocking agent (1-5) with a slight @ stimulating effect (6). I t was introduced in 1950 ( 7 ) as a vasodilating drug for intravenous and oral administration (8). I t has been reported as a useful agent for therapy in congestive heart failure, myocardial infarction, arrhythmia, angina pectoris, shock, and bronchial asthma (9). Current pharmacologic investigations of phentolamine have been summarized in t h e proceedings of a symposium (10). Because of t h e recent interest for use of this drug as a continuous infusion or for long-term therapy, a simple, specific, quantitative assay in biological media is desirable. Techniques applied to t h e analysis of pharmaceutical preparations include titrimetry ( 2 ) , gravimetry ( I ) , UV spectrophotometry ( 1I), colorimetry (12-14), gas chromatography (15),and high pressure liquid chromatography (16). T h e colorimetric reaction (17) has been adapted for use in biological samples (18),but was not found to be specific for phentolamine. T h e H P L C analysis of phentolamine utilizes an ion-exchange column (16);however, the method is characterized by asymmetric peaks and poor resolution ( R < 1.0). Analysis of imidazoline compounds in biological samples with T L C (19) is specific for phentolamine, but is limited to quantities greater than 1 pg. In addition, the TLC technique 'Present address, Astra Pharmaceutical Products, Inc., Framingham, Mass. 01701. 0003-2700/78/0350-0521$01.OO/O
Reagents and Solvents. Phentolamine mesylate was provided by Ciba-Geigy, Summit, N.J. Antazoline base and naphazoline hydrochloride were obtained from Pfaltz and Bauer, Stanford, Conn. Sodium octane sulfonic acid was purchased from Eastman Chemicals, Rochester, N.Y. Triple distilled methanol, diethyl ether, and cyclohexane were obtained from Burdick and Jackson Laboratories, Muskegon, Mich. Distilled, deionized, neutrai, charcoal filtered, and bacteria free water was used for all solutions. All other reagents were analytical grade or better. Apparatus. A liquid chromatograph with a Milton Roy Minipump (Milton Roy Company, Riviera Beach, Fla.) was used. A pulseless solvent flow was obtained with a T configuration of dampers and restrictors (Waters Associates, Milford, Mass.). Pressure was monitored continuously with a glycerine filled, 5000 psig Lenz gauge through a high pressure manifold connected to a 10000 psig Circle Seal adjustable relief valve, set for a 5000-psig cracking pressure. The mobile phase was continuously filtered through the solvent inlet line by a 30-pm filter (No. 25531, Waters Associates). Further particulate matter was removed from the solvent by a 2- and 0.5-pm filter (Swagelok SS2F-2 and SS2000-SR12, respectively) placed in series after the pulse damping network. Samples were injected through a 7OOO-psig, six-port Valco valve, with a 50-pL loop. A Glenco sample injection syringe (Model VIS 50-700) and filling port (VISF-1) were used. Separations were accomplished on a Microbondapak/CI8 column (U'aters Associates) maintained at constant temperature in a 20-L water bath. The drug concentration in the colunin effluent was monitored by a variable wavelength detector (model SF770, Schoeffel Instruments. Westwood, N.J.). Chromatograms were recorded with a model 252A Linear Instruments strip chart recorder. Chromatographic Conditions. Separations were performed at 2.4 mL/min and at a back pressure of 4000 psig. The column 1978 American Chemical
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