Anal. Chem. 1989, 67, 566-570
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Salicylate-Selective Membrane Electrode Based on Tin( 1V) Tetraphenylporphyrin N. A. Chaniotakis, S. B. Park, and M. E. Meyerhoff* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055
The response propertles of a new solvent/polymerlc membrane electrode with unique Selectivity toward anionic salicylate are reported. The electrode is prepared by lncorporating 5,10,15,20-tetraphenyl(porphyrIr;ato)tln( I V ) dichloride (Sn[TPP]CI,) Into a plasticized poly( vinyl chloride) membrane. The resultlng sensor exhibits an anti-Hofmelster selectivity pattern, with high specificity for salicylate over lipophilic inorganic anions (perchlorate, periodate, thiocyanate, Iodide, etc.) and biological organic anions (citrate, lactate, acetate). Moderate Selectivity over structural analogues of salicylate (3and 4-hydroxybenroate, benzoate) is also observed. Radiotracer uptake experiments using [“C]salicylate clearly show that the metal center of the metalloporphyrin is critical for selective salicylate transport In the membrane phase. Minimal response to chloride ions makes the new electrode potentially useful for estimating salicylate levels in biological samples.
INTRODUCTION Recently, efforts to devise new anion-selective electrodes have focused on the use of organometallic species and metal-ligand complexes as membrane active components (1-3). When these reagents are incorporated into plasticized poly(vinyl chloride) (PVC) membranes, the resulting potentiometric sensors can display selectivity sequences that are markedly different than those observed when membranes are doped with classical anion exchangers (e.g., quaternary ammonium species). Metalloporphyrins are one class of molecules which, in preliminary studies ( 4 , 5 ) ,have proven useful for such purposes. Indeed, it has been shown previously that anion selectivities of manganese(II1) porphyrin based membranes can be altered by changing the peripheral structure of porphine ring surrounding the central metal (6). When relatively unhindered manganese(111) tetraphenylporphyrin (Mn[TPP]Cl) was utilized as the active membrane component, marginal selectivity for organic salicylate over many inorganic anions was observed (6). The present work demonstrates how merely changing the metal center in TPP from Mn(II1) to Sn(IV) dramatically increases the selectivity toward salicylate. Salicylate and its analogues, including acetylsalicylate (aspirin), are commonly used as effective analgesics and are available to the public in a wide variety of formulations. Despite their utility as pain relievers and antipyretics, salicylates can be quite toxic if taken in large doses. Recommended therapeutic levels in plasma range from 0.15 to 2.1 mmol/L (total salicylate), although a large fraction of this total salicylate may be bound to proteins ( 7 , 8 ) . The most widely used analytical method for determining totalsalicylate is based on the Trinder reaction in which sample salicylate reacts with ferric ions to form a colored complex in acid solution (9). Selectivity of this method is poor, since similar complexes can form with a variety of compounds, including endogenous phenolic compounds (e.g. tyrosine), enolic metabolites (e.g. acetoacetate), and certain drugs (e.g. phenothiazenes). Per-
* Author
to whom all correspondencesshould be addressed
haps more importantly, the Trinder method, as well as some of the newer salicylate measurement techniques (e.g., enzymatic ( l o ) ,chromatographic ( 1 1 , 12)) measure “total”, not “free”, salicylate although it is known that the latter correlates more closely to the drug’s biological activity (13) (note: “free” being defined as unbound salicylate concentration in undiluted samples a t physiological pH). The development of a suitable salicylate-selective membrane electrode would enable the detection of “free” salicylate activity in samples and could aid researchers in studying the pharmacological role of this drug. Moreover, positive results from recent studies regarding the effect of aspirin on preventing heart attacks ( 1 4 ) suggest that a much larger number of individuals will be taking this drug in the future. This will mandate improved salicylate detection methods, perhaps ones that allow for measurement in undiluted whole blood. Polymeric membrane electrodes for salicylate measurements have been proposed previously (15,16). However, since these earlier probes were based on quaternary ammonium salts of salicylate as dissociated ion-exchange type membrane components, these electrodes lacked adequate selectivity over a number of common physiological anions (e.g., chloride and lactate) to be applied for measurements of salicylate in biological samples. It will be shown that the new metalloporphyrin-based salicylate sensor described herein offers a substantial improvement in selectivity over these anions, rendering this membrane electrode potentially useful for measuring salicylate in physiological samples.
EXPERIMENTAL SECTION Reagents. For all the potentiometric measurements, 2morpholinoethanesulfonic acid (MES) (Aldrich Chemical Co., Milwaukee, WI) was used to buffer the sample or test solutions. The carrier, 5,10,15,20-tetraphenyl(porphyrinato)tin(IV) dichloride (Sn[TPP]C12)was synthesized from 5,10,15,20-tetraphenyl21H,23H-porphine [TPP]H2(Porphyrin Products, Logan, UT), and SnCl,, according to methods described elsewhere ( 1 7). Dibutyl sebacate (DBS) was obtained from Kodak (Rochester, NY) and used without any further purification. Poly(viny1chloride) (PVC), chromatographic grade, was a product of Polysciences, Inc. (Warrington, PA). For the radioactivity uptake experiments, [7-’*C]salicylicacid (New England Nuclear, Boston, MA) was used. Organic and aqueous scintillation cocktails were obtained from RPI (Mt. Prospect, IL). Apparatus. Cell potentials were measured at 25 “C. For most experiments, the following galvanic cell was employed: Hg/ Hg,Cl,(s), KCl (saturated)/MES (0.05 mol/L), KCl (0.1 mol/L) Nasal (0.0001mol/L), pH 5.5/sample solution/PVC-porphyrin membrane/MES (0.05 mol/L), KC1 (0.1 mol/L), Nasal (0.0001 mol/L), pH 5.5, AgCl(s)/Ag. The external reference electrode was a double junction cracked-bead SCE electrode although accurate measurements can be made with a single junction reference owing to the high selectivity of the Sn[TPP]Cl,-based membranes for salicylate over chloride. The electrodeswere connected through a high impedance amplifier to a Zenith 2-100 PC computer, equipped with an analog to digital converter (DT2801, Data Translation, Inc., Malborough, MA). The polymeric membrane composition was typically 1 w t 70 porphyrin carrier (Sn[TPP]C&),66 wt % DBS, and 33 wt % PVC. All membranes were cast as described previously ( I ) . The membranes were c u t from the cast films with a 7 2 cork cutter,
0003-2700/89/0361-0566$01.50/0 1 1989 American Chemical Society
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and mounted within Phillips electrode bodies (ISE-561) (Glasblaserei Moller, Zurich). Determination of emf Response and Selectivity of the Membranes. Anion selectivity coefficients were determined by the separate solution method, except for hydroxide interference which was estimated by the fixed interferent method (18). Concentrations were used rather than activities due to uncertainties in estimating ionic strengths of zwitterion background electrolytes. The sample solution electrolyte was 0.05 mol/L MES buffer, adjusted to pH 5.5 by small additions of NaOH solution. Concentrations of weak acid anions in the test solutions were calculated based on the fraction of the ionized form present at pH 5.5 in accordance with the Henderson-Hasselbalch equation. The response of the salicylate-membrane electrode toward pH was obtained by titrating 0.05 mol/L phosphoric acid with small aliquots of NaOH, while simultaneously monitoring the pH of the sample solution with a combination glass pH electrode. Radiotracer Experiments. The uptake of [7-14C]salicylate by various polymeric membranes was determined as follows: Small disks of the appropriate membranes were cut and placed into 200 NLof a 0.30 mmol/L solution of salicylate composed of 6% hot and 94% cold sodium salicylate (prepared in MES buffer, pH 5.5). After being shaken for a preset time period, the membranes were removed, washed briefly with MES buffer, pH 5.5, and then dissolved in 0.5 mL of tetrahydrofuran. The dissolved membrane solution was mixed with 5 mL of organic scintillation cocktail and the radioactivity in the resulting mixture was counted with a scintillation counter (Beckman-LS 3801). Serum and Urine Salicylate Studies. SeraChem Level 1 Clinical Chemistry Control Human Serum-Assayed (Fisher Blood Chemistry Controls) was used for the serum recovery study. Two milliliters of the serum was reconstituted with distilled water, pooled, and then filtered through a 20000 molecular weight cutoff membrane (Micro Filtration Systems, Dublin, CA), to remove proteins. Samples were then prepared from this albumin-free serum by spiking with different levels of sodium salicylate. At this point, 1 mL of each spiked solution was separated and sent to the clinical analysis laboratory (University of Michigan Hopsitals) for colorimetric salicylate determinations on a Du Pont ACA instrument. The remainder of the solution was diluted (1:lO) with 0.05 mol/L MES buffer, pH 5.5, for emf measurements. The calibration curves used for this set of experiments were obtained by using salicylate standards prepared in 0.15 mol/L saline and treated the same way as the filtered serum samples. After the electrodes were allowed to equilibrate in saline-MES buffer solution with no added salicylate, the sample solutions were assayed in random order. A similar procedure was used to perform recovery and correlation studies on human urine samples, except that no prior ultrafiltration procedure was required due to the low protein content of urine. R E S U L T S A N D DISCUSSION Selectivity and Response C h a r a c t e r i s t i c s of Sn[TPP]C12-Based Membrane Electrode. The potentiometric anion selectivity coefficients obtained with the new Sn[TPP]C12-basedPVC membrane electrode are listed in Table I. For comparison purposes, selectivity data is also presented for a previously reported salicylate electrode (based on Aliquat-336) (15) and membranes prepared with Mn[TPP]C1(6), all under the same experimental conditions (in background of MES buffer, pH 5.5). It is clear that the electrodes prepared with the metalloporphyrins exhibit selectivity sequences that are rather different than the Hofmeister pattern found with the Aliquat-based membrane. Moreover, with the exchange of Sn(1V) for Mn(II1) as the central metal of TPP, a further increase in response toward salicylate relative to many of the other anions tested is observed. Of particular significance is the fact that selectivity over several lipophilic anions (perchlorate, thiocyanate, periodate, etc.) is enhanced nearly 1000-fold,while selectivity for salicylate over chloride is nearly 100 times greater. In previous work ( 4 ) , lack of adequate selectivity over chloride precluded use of the Mp[TPP]Cl based membranes for direct measurements in biological sam-
Table I. Selectivity Coefficients, log l&=, of Salicylate Electrodes Prepared with Different Membrane Active Componentsa Aliquat 336-Salb
anion
salicylate
Sn [TPP]C1ze
0.0 -2.1 -1.4
c1-
BrI-
IO4c10,SCN' salicylurate benzoate citrate acetate lactate m-hydroxybenzoate p -hydroxybenzoate
0.0 -2.1
0.0
-3.8 -3.7 -3.7 -3.6 -3.4 -2.5 -1.5
0.1 1.9 1.8 0.7 -1.1 -1.2
Mn[TPP] Cld
-1.8 -0.6 -0.3
-0.6 -0.8
-0.5
-1.4
-1.8
-3.8 -3.9 -4.0
-2.2 -2.2 -1.4
-1.1 -1.0
-1.3
"All values obtained in 0.05 mol/L MES, pH 5.5, at an anion concentration of 0.01 mol/L. * Aliquat-336s membranes were prepared with 63 wt % n-dibutyl phthalate, 30 wt % PVC and 7 wt 70 Aliquat. Sn[TPP]Clz-basedmembranes were prepared with 66 wt % DBS, 33 w t % PVC, and 1 wt % carrier. dFrom ref 6. 120
-
100
-
60
-
40
-
20
-
80
>
E
0-20 -40
I
I
I
I
-6
-5
-4
-3
-2
Log[Salicylate], M
Figure 1. Potentiometric salicylate response of PVC membranes doped with varying levels of Sn[TPP]CI,: (0)1 wt % of porphyrin; (W) 0.3 wt % of porphyrin; (+) 0.1 wt % of porphyrin. Measurements were made in 0.05 mol/L MES buffer, pH 5.5.
ples. In fact, selective measurement of salicylate in the presence of high chloride could only be made after acidifying the sample and allowing neutral salicylic acid t o permeate a silicone rubber membrane ( 4 ) . The marked increase in salicylate selectivity using the tin(1V) porphyrin should obviate the need for such selectivity enhancement techniques (i.e., use of additional membranes). As with many carrier-mediated membrane electrodes, the total potentiometric response of the electrode toward salicylate is dependent on the concentration of the metalloporphyrin incorporated within the membrane. As shown in Figure 1, increasing levels of porphyrin results in membranes that display larger slopes and lower limits of detection. Using 1 w t % of Sn[TPP]Cl, in the membrane yielded electrodes with near-Nernstian response toward salicylate (55-60 mV/decade), over a wide range of salicylate concentrations. At concentrations above 1 wt 70,unusual super-Nernstian behavior is observed. However, a t these higher concentrations, the solubility of the Sn[TPP]C12in the membrane is exceeded and this results in a rather heterogeneous and irreproducible membrane phase. The response times of porphyrin-based membranes are somewhat slower than those observed with classical ion-exchanger or neutral carrier-type membrane electrodes. In the
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989 70
> E
-4
Log[Salicylate], M Figure 2. Response time (90% of equilibrium response) of new salicylate electrode as a function of sample salicylate concentration in MES buffer, pH 5.5. 230
-3 8
- ‘8
-2 8
Log[Salicylate], M Figure 4. Salicylate response of Sn[TPP]CI,-based membrane electrode in 0.05 mol/L MES buffer at various pH values: ( 0 )pH 5.5; (0) pH 6.2; (+) pH 7.2.
10
1
I
8
9 8
-120
-’O
I
i 1 0
t
3 J1
c
30
50
70
90
110
’20
PH
Figure 3. pH response of Sn [TPP] &based membrane electrode. case of the Sn[TPP]C12electrode, response times (90% of equilibrium response) range from 2 to 10 min, depending on concentration of salicylate in the sample (see Figure 2). Surprisingly, unlike most membrane electrodes, response was more rapid at very low and very high salicylate levels, and somewhat longer at millimole per liter concentrations. This unusual behavior may be related to the fact that the Sn[TPP]C12carrier has two possible sites for ligand (anion) exchange, and simultaneous equilibria involving both of these sites may only occur as the anion (salicylate) concentration in the sample falls within a finite range (see later section on mechanism). P r o t o n Response of Tin(1V) P o r p h y r i n Membrane. Figure 3 shows the pH response of the Sn[TPP]Cl,-based membrane evaluated by titrating 0.05 mol/L phosphoric acid with NaOH. Similar potentiometric pH responses have been observed previously with manganese(II1) porphyrins and have been ascribed to the coordination of water molecules as axial ligands to the central metal (6). Once associated, the water can readily lose a proton to the bathing sample solution, yielding a hydroxide-coordinated metal that results in a response analogous to that resulting from anion coordination at the same axial ligand site. This mechanism was supported by recent studies regarding the effects of using aminated and carboxylated PVC (19) in preparing ion-selective electrodes. In that work, the Sn[TPP]C12served as a model anion selective carrier. The analytical implications of the pH responsiveness of the new salicylate selective membrane electrode can be gauged more accurately by examining the response toward salicylate in the same buffer system, but at different pH values. As shown in Figure 4, detection limits toward salicylate deteriorate as the pH of the buffer increases. (Note: at pH values
0
20
40
60
80
100
Time, min. Figure 5. [ 14C]Salicylate uptake of PVC membranes as a function of time: (e)blank-PVC; (+) [TPP]H,-PVC; (W) Sn[TPP]CI,-PVC. Insert: Radioactivity taken up by Sn[TPP]GI,-PVC membranes plotted as a function of the square root of time. below 5.5 a significant fraction of salicylate becomes protonated a form not detected by the electrode.) The observed behavior is not due to higher concentrations of anionic MES in the pH 6.2 and 7 . 2 buffers. Indeed, in separate experiments (at constant pH), it was found that the membrane exhibits little or no response to anionic MES. Thus, the data shown in Figure 4 can be used to calculate an effective selectivity coefficient for the salicylate relative to hydroxide ion using sal OHthe fixed interferent method. The value calculated, log kPo: = 4.2 (at pH 7.2), limits the practical applications of this new salicylate sensor in monitoring “free” salicylate activity under physiological conditions (pH 7.2-7.6). Under these conditions, therapeutic free salicylate levels would be masked by the apparent hydroxide response of the sensor (see Figure 4). Efforts aimed at decreasing the pH response and, thus, the apparent selectivity coefficient relative to hydroxide ions are currently in progress. Mechanism of Salicylate Response a n d Selectivity. The unique potentiometric selectivity sequence of the Sn[TPP]Cl,-based membrane must be related to the ability of the membrane to partition and transport salicylate preferentially over other anionic species. To corroborate the observed potentiometric results and further prove that the Sn(IV) center in the metalloporphyrin is important for inducing salicylate selectivity, radiotracer uptake experiments were performed by using [7-14C]salicylateas described in the Experimental Section. Figure 5 shows the amount of radioactive salicylate incorporated into PVC membranes as a function of contact time. As can be seen, there is very little partitioning of salicylate into blank PVC membranes or ones prepared with
ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989
Table 11. Recovery of Salicylate Added to Protein-Free Seruma and Human Urine
salicylate added mmol/L 0.00
0.64 0.99 1.60
0.00
0.79 1.24 1.99
salicylate found, mmol/L electrode* colorimetricC
av %
recovery
105 104 96.2
urine 0.23 f 0.01 0.98 f 0.03 1.44 f 0.10 2.27 f 0.10
94.9 97.6 103
*
Table 111. Comparison of Results Obtained for Measurement of Salicylate Concentrations in Spiked and Nonspiked Human Urine Samples by Membrane Electrode and Colorimetric Methods
(electrode)
serum 0.10 f 0.01
