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(16) Gough, D. A.; Leypold, J. J . Electrochem. SOC. 1980, 127. 1278. (17) Mancy, K. H.; Okun, D. A.; Reilly, C. N. J . Nectroanal. Chem. 1962, 4 , 65. (18) Schulmeister, T.; Scheller, F. Anal. Chim. Acta 1985, 170,279. (19) Rucicka, J.; Hansen, E. Anal. Chim. Acta 1978, 99,37. (20) Reijn, J. M.; van der Linden, W. E.; Poppe, H. Anal. Chlm, Acta 1980, 114,105. (21) Vanderslice, T. J.; Stewart, K. K.; Rosenfeld, A. G.; Higgs, D. J. Talanta 1981, 28, 11. (22) Reijn, J. M.; van der Linden, W. E.;Poppe, H. Anal. Chim. Acta 1981, 126, 1. (23) Rucicka, J.; Hansen, E. "Flow Injection Analysis"; Wiley: New York, 1981; Chapter 3. (24) Poppe, H. Anal. Chim. Acta 1980, 114, 59. (25) Macholan, L.; Londyn, P.; Fischer, J. Collect. Czech. Chem. Commun. 1981, 4 6 , 2871. (26) Pacakova, V.; Stulik, K.; Brabcova, D.; Barthova, J. Anal. Chim. Acta 1984. 159. 71. (27) Nentwig, J:; Scheller, F.; Weise, H.; Pfeiffer, D. DD Patent, GOIN 2 770 084. 1905. (28) Scheiier, F.; Pfeiffer, D.; Seyer, I.; Kirstein, D.; Schulmeister, T.; Nentwlg, J. Bioelectrochem. Bioenerg. 1983, 1 7 , 155. (29) Crank, J. "The Mathematics of Dlffusion", 2nd ed.; Claredon Press: Oxford, 1975; p 49.
(30) Levenspiel, 0. "Chemical Reactlon Engineering", 2nd ed.; Wiley: New York, 1972; p 258. (31) Feldberg, S. W. I n "Electroanalytical Chemistry"; Bard, A. J., Ed.; Marcel Dekker: New York, 1969; Vol. 3, Chapter 4. (32) Satterfield, C. N. "Heterogeneous Catalysis"; M.I.T. Press: Cambridge, MA, 1970; p 135. (33) Annino, R. I n "Advances in Chromatography"; Giddings, J. C., Grushka, Cazes and Brown, Eds.; Marcel Dekker: New York, 1977; Vol. 15, Chapter 2. (34) Sternberg, J. C. I n "Advances in Chromatography"; Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker: New York, 1966; Vol. 2, Chapter 6. (35) Arbramowitz, M.; Stegun, I. D. "Handbook of Mathematical Functions (AMs 55)";National Bureau of Standards: Washington DC, U.S. Government Printing Office, 1964. (36) Ginzburg, R. 2.; Katchalsky, A. J . Gen. Physiol. 1963, 47,403. (37) Swoboda, B. E. P.; Massey, V. J . Biol. Chem. 1965, 240, 2209.
RECEIVED for review June 27,1985. Resubmitted November 25, 1985. Accepted December 2, 1985. This work was made as a part of a cooperation supported by the Academy of Sciences of GDR and the Royal Swedish Academy of Sciences.
New Liquid Membrane Electrode for the Determination of Ephedrine, Epinephrine, and Norepinephrine Saad S. M. Hassan' and G . A. Rechnitz*
Department of Chemistry, University of Delaware, Newark, Delaware 19716
A new liquid membrane electrode based on an Ion assoclatlon extraction system responding to ephedrlne Is described. I t incorporates an ephedrlne-flavianate ion palr complex as a novel electroactive component in 1-octanol. The electrode exhibits near-Nernstian response over the concentration range of lo-* to M ephedrlne In soiutlons of pH 4-7. The electrode also responds to some neurotransmitterscontalnlng the 6-ethanolamine moiety. Response tlme varies from 20 to 90 s, and interferences from many organic bases and some common lnorganlc cations are negliglble. Determinations of 0.1-2000 pg/mL ephedrlne, epinephrlne, and norepinephrine give results wlth an average recovery of 98.7% and a mean standard deviation of 2.3 %.
Ephedrine, epinephrine, and norepinephrine are aryl-@ethanolamine derivatives having characteristic physiological function and pharmacological action. Ephedrine and epinephrine stimulate both a and fl adrenergic receptors, whereas norepinephrine acts only on the a receptors. Ephedrine is used as a drug in therapeutic doses a t the level of 15-60 mg to produce peripheral vasoconstriction, to raise blood pressure, to prevent hypotension, and to treat allergic states, catalepsy, and myasthenia gravis. Epinephrine and norepinephrine, on the other hand, are produced in mitochondria and are released in microgram quantities from the peripheral endings of the sympathetic nerves when this part of nervous system is stimulated (1, 2). Methods commonly used for the determination of these sympathomimetic m i n e s in pharmaceutical preparations and biological samples are mainly based on extraction followed by spectrophotometric and spectrofluorometric measurements (3, 4). Present address: Qatar University, Faculty of Science, Doha, Qatar.
Ephedrine and most catecholamines are spectrophotometrically measured a t 241 and 279 nm, respectively (2, 4 ) . Epinephrine and norepinephrine are determined by methods based on their oxidation to adrenochromes, which rearrange in alkaline media to the fluorescent lutins, followed by fluorescence measurement at 400-505 mm (2,3). Methodological complications and deficiencies are commonly encountered in using these methods. Many organic compounds and drug excipients absorb substantially in the ultraviolet region of the spectrum and thus interfere in the spectrophotometric procedure. The fluorescent lutins are unstable and must be protected from oxidation and quenching substances. Membrane electrodes incorporating ephedrine-tetraphenylborate dispersed in organic solvents or poly(viny1chloride) matrices have recently been used for direct potentiometric monitoring and potentiometric titration of ephedrine (5-9). These sensors, however, suffer from severe interferences by various classes of amines, some inorganic cations, and many of the excipients commonly used in drug formulations. In the present work, we describe a new liquid membrane electrode with significantly improved characteristics for ephedrine. It is based on the use of ephedrine-flavianate as a novel electroactive material. The electrode yields good response to the neurotansmitters epinephrine and norepinephrine and has negligible interference from many organic and inorganic bases.
EXPERIMENTAL SECTION Reagents. All solutions were prepared with deionized, doubly distilled water and reagent grade chemicals, except where otherwise stated. Flavianic acid (2,4-dinitro-l-naphthol-7-sulfonic acid), ephedrine hydrochloride, epinephrine hydrochloride, norepinephrine hydrochloride,dopamine hydrochloride,DL-dopa, tyrosine, and all other amines were obtained from Sigma Chemical Co. (St. Louis, MO). Fresh solutions were prepared daily and protected from light. Membrane Preparation. The ephedrine-flavianate complex was prepared by mixing and stirring a 20-mL aliquot of lo-' M
0003-2700/86/0350-1052$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table I. Response Characteristics of Ephedrine-Flavianate Liquid Membrane Electrode at 25 "C in Some Lipophilic Solvents parameter slope, mV/(log C ) std dev, mV corr coeff ( r ) intercept, mV
lower limit of linear range, M
nitro1-octanol 1-decanol benzene 55.2 0.7
0.9996 208.2
46.5 0.9 0.9997 131.0 10-4
53.0 0.8 0.9992 157.5 10-4
ephedrine hydrochloride solution with a 30-mL aliquot of lo-' M flavianic acid solution. The precipitate was filtered with a G-3 sintered glass crucible, washed with deionized, doubly distilled water followed by ethanol, dried at 100 "C for 1 h, and ground to a fine powder. A M ephedrine-flavianate solution in 1-octanol was used as the liquid membrane material. Electrode Assembly. An Orion membrane electrode body (Model 92) equipped with an Orion 92-05-04 porous membrane was assembled as previously described (IO). A lo-' M ephedrine-flavianate solution in 1-octanol and an aqueous solution containing M of both ephedrine hydrochloride and potassium chloride were used as liquid membrane and internal reference solution, respectively. The electrode was conditioned for 24 h by soaking in M aqueous ephedrine hydrochloride solution and was stored in deionized, doubly distilled water when not in use. Potential Measurements. Potential measurements were made with the ephedrine-flavianate liquid membrane electrodes in conjunction with a double junction Ag/AgCl reference electrode (Orion Model 90-02) containing 10% KN03 in the outer compartment. The electrode potential was measured at 25 f 0.5 "C with an Orion microprocessor ionalyzer (Model 901). Adjustment of pH was carried out by using an Orion combined glass-calomel electrode system (Model 90-00). Electrode Calibration. The ephedrine-flavianate liquid membrane electrode in conjunction with the reference electrode was immersed in 15-mL aliquots of to lo4 M ephedrine, epinephrine, and norepinephrine hydrochloride solutions. The pH was adjusted to 4-7 with dilute KOH; the potential readings were recorded after stabilization to f0.5 mV and plotted as a function of the logarithm of ephedrine, epinephrine, and norepinephrine concentration. The graphs were used for subsequent determination of ephedrine, epinephrine, and norepinephrine in unknown samples. Electrode Selectivity. The selectivity coefficients were evaluated based on the potential measurement in mixed solutions that contained M ephedrine hydrochloride solution and from lo-' to low5M of the interfering compound. The selectivity coefficients, KA pot, were calculated according to the relation where A E is the change in poKA,$"~= (lohE/'- l)~A/(uB)l/**, tential in the presence of the interfering compound B whose charge is either z+ or z- for cationic or anionic forming species, respectively, S is the slope of the calibration graph for ephedrine, and UA and UB are the concentations of protonated ephedrine and the interfering compound, respectively. R E S U L T S AND DISCUSSION Membrane Material a n d Characteristics. It has been reported that some organic bases form crystalline precipitates with flavianic acid (11). In this study, ephedrine-flavianate was prepared, characterized, and tested as a novel ion exchanger site in a liquid membrane electrode responsive for ephedrine. Liquid membrane electrodes consisting of ephedrine-flavianate dissolved in liphophilic solvents such as 1-octanol, 1-decanol, and nitrobenzene were prepared. The response characteristics of these electrodes were evaluated at 25 f 0.5 "C over the course of 30 days for standard solutions of ephedrine hydrochloride. Linear regression analysis of data obtained is given in Table I. It can be seen that these electrodes display linear response of nearly Nernstian character in ephedrine solutions of conM. This indicates the centrations down to approximately
70
1053
I
e
I
1
I
I
5
4
3
2
-Log
[EphedrlndvM
Flgure 1. Calibration plots for ephedrine-flavianate liquid membrane electrodes with (0)1-octanol, (0)nitrobenzene, and (0) ldecanol as membrane solvents.
feasibility of using these electrodes for the determination of low levels of ephedrine. The lower limit of linear response and slope of the calibration plots are affected by the nature of the solvent (Figure 1). 1-Octanol provides a wide linear response, stable potential readings, and low limit of detection. All the results shown below are obtained with this electrode. The slope of the linear part of the curve is 55.2 f 0.6 mV/ concentration decade. The slope is stable within f 2 mV/log [ephedrine] up to a period of 4 weeks. During 1 day's use, the potential readings vary by not more than f l mV, and the drift over a period of 1week is about f3 mV. Liquid memor mol L-l ephedrine-flavianate exhibit branes with almost the same characterization. The poor sensitivity offered by membrane electrodes incorporating 1-decanol or nitrobenzene (Figure 1)solvents is due to the poor solubility of the ephedrine-flavianate ion pair complex in these solvents. We found that the electrode also responds to epinephrine and norepinphrine cations. These compounds are structurally similar to ephedrine, being aryl-p-ethanolamine derivatives. The electrode exhibits linear potential response in the range M norfrom to M epinephrine and to epinephrine with cationic slopes of 45.1 and 35.2 mV/concentration decade, respectively. Calibration data yield least-squares equations of (AE, mV) = (45.1 f 0.1) log C (162 f 0.3) and (AE, mV) = (35 f 0.2) log C + (80 f 0.3) for epinephrine and norepinephrine, respectively. The standard error is 0.8 mV. The potential readings and slopes are almost constant over 3 weeks. Attempts to prepare electrodes incorporating epinephrine- and norepinephrine-flavianate ion pair complexes were unsuccessful probably due to weak complexation tendency and/or low solubility of the complexes in the organic solvents. Response Time. The times required for the ephedrineflavianate liquid membrane electrode to provide stable emf readings within f l mV of the steady potential by either 10fold increase of ephedrine, epinephrine, and norepinephrine concentrations to the same solutions or after successive immersion of the electrode in a series of ephedrine, epinephrine, and norepinphrine hydrochloride solutions each having a 10-fold concentration difference were measured. Both ex-
+
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table 11. Selectivity Coefficients for Ephedrine-Flavianate Liquid Membrane Electrode interfering species (B)
100-
m e t h y l urea piperidine glycine dimethylamine succinamide epinephrine norepinephrine
80 -
>
5
60-
c C
0 *
D"
4.0 X 3.5 X 1.1 X 1.7 X 1.4 X 9.9 X 9.7 X
lo-' lo-' lo-' lo-' lo-' lo-'
oxalic acid citric acid
NH4+
2.3 X 1.7 X 3.7 x 10-3
Kt
4.3 x 10-3 3.2 x 10-3
BaZt
1.1 x 10-3
Nat
40-
U 0
0
L
;+20-
W
selectivity coeff interfering selectivity coeff (KA,B"~) species (B) (KA,BPat)
'
2
4
6
8
10
PH
Figure 2. Effect of pH on the potential of ephedrine-fiavianate liquid membrane electrode.
periments show short response times, normally 20-30 s for solutions 210-3 and 1.5 min for solutions M of these compounds. Electrode age of up to 20 days has no effect on the response time. The electrode can be used for 25 days before renewing the membrane and without any noticeable deterioration in the response. Effect of pH. The pH dependence of the ephedrine electrode is shown in Figure 2 a t three different ephedrine concentration levels. It can be seen that electrode response is independent of pH over the 4-7 pH range and that the region of pH independence is mildly concentration dependent. The significant increase of the potential observed below pH 4 may be due to interference by H+, and the decrease of the potential above pH 8 is probably due to the increase in the concentration of the unprotonated amines or the precipitation of the free base. Effect of Interfering Compounds. The effects of high background levels of some basic, acidic, and neutral compounds as well as inorganic anions and cations were assessed. Selectivity coefficients values (KA,B)determined by the standard mixed solution method (IO)are listed in Table 11. These results show that the ephedrine-flavianate liquid membrane electrode has a reasonable selectivity for ephedrine, epinephrine, and norepinephrine and exhibits negligible interferences from many amines, amides, amino acids, carboxylic acids, and common inorganic cations. Compared to other ephedrine membrane electrodes, the present electrode system exhibits higher selectivity toward many amines and inorganic cations (9). Some pharmaceutical diluents and excipients normally used in drugs and compounds normally associated with the neurotransmitters are also examined for their effect on the electrode response. No interference is measurable for
maltose, glucose, lactose, Tween-80, and starch a t levels as high as M. Tyrosine, dopa, and dopamine interfere a t levels LW3M. Determination of Ephedrine, Epinephrine, and Norepinephrine. The results obtained for the determination of 0.1-2000 kg/mL ephedrine, epinephrine, and norepinephrine hydrochlorides in aqueous solutions using the ephedrineflavinate liquid membrane electrode with the calibration graph method show an average recovery of 98.7% and a mean standard deviation of 2.3%. Thus, the new electrode may provide a rapid, sensitive, and inexpensive method for the determination of some biogenic amines with minimal sample pretreatment. The low detection limit offerd by the electrode along with the reasonable selectivity and stability may enhance its practicality in pharmaceutical and biochemical analyses. This was demonstrated by determination of ephedrine in some pharmaceutical injections, tablets, syrups, and eye drops containing 2-50 mg of ephedrine/mL or tablet. A portion equivalent to 25-75 mg of ephedrine was treated with 15 mL of 0.05 N HCl, heated to 60 O C for 3 min, and the pH adjusted to 4-7. The solution was then diluted to 25 mL with deionized, distilled water and shaken. The potential of the solution was then measured with the electrode system and compared with the calibration graph. The results obtained show an average recovery of 97% of the nominal values, and the mean standard deviation is 2.5%.
LITERATURE CITED (1) Williams, R. J. "The Encyclopedia of Biochemistry"; Lansford, E. M., Jr., Ed.; Reinhold: New York, 1967;pp 17-18. (2) Nagatsu, T. "Biochemistry of Catecholamines"; University Park Press: Baltimore, MD, 1973;pp 209-273. (3) Tietz, N. W., Ed. "Fundamentals of Clinical Chemistry"; Saunders: Philadelphia, PA, 1970;pp 569-586. (4) British Pharmaceutical Codex, BPC, Pharmaceutical Press: London, 1973;pp 178-179. (5) Selinger, K.; Staroscik, R. Pharmazie 1978, 3 3 , 208-212; Chem. Abstr. 1978, 89,9 5 0 5 5 ~ . (6) Fukamachi, K.; Nakagawa, R.; Morimoto, M.; Ishibashi, N. Bunseki Kagaku 1975, 2 4 , 428-432;Chem. Abstr. 1975, 83, 168515j'. (7) Goina, T.; Hobai, S.; Rozenberg, L. Farmacia (Bucharest) 1978, 2 6 , 141-147;Chem. Abstr. 1979, 90,76618m. (8) Zeng, J. Yaoxue Xuebao 1982, 1 7 , 841-846; Chem. Abstr. 1983, 98,95746h. (9) Selinger, K.; Staroscik, R. Chem. Anal. (Warsaw) 1982, 2 7 , 223237; Chem. Abstr. 1983, 99, 110842~. (10) Ma, T. S.;Hassan, S. S. M. "Organic Analysis Using Ion Selective Electrodes"; Academic Press: London, 1982;Vol. I. (11) Wachsmuth, H. J . Pharm. Belg. 1953, 8 ,283-288.
RECEIVED for review September 9,1985. Accepted December 23, 1985. We gratefully acknowledge the financial upport of Grant GM-25308 from the National Institutes of Health.
