tended the conversion of L-leucine into its N-TFA-(+)-2butyl ester during the customary 3-hour reflux period, and that only negligible (less than 1%) racemization occurred even after 23 hours.
RECEIVEDfor review May 31, 1974. Accepted August 14, 1974. We are indebted to the Nationai Aeronautics and Space Administration for a Research Grant (No. NGL-05020-582) which supported a portion of this work.
Determination of Morphine in Biologic Fluids by Electron Capture Gas-Liquid Chromatography Jack
E. Wallace and Horace E. Hamilton
Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Kenneth Blum Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Clayton Petty Division of Anesthesiology, University of Arizona College of Medicine, Tucson, Ariz. 8572 1
A method that permits the quantitative determination of morphine at therapeutic levels in 1-2 mi of serum or plasma is described. Morphine levels at less than twenty-five nanograms per mi can be effectively assayed providing an internal standard of nalorphine is employed. Both the opiates are measured by electron capture detection (63Ni) as their respective trifluoroacetyl derivatives. Sensitivity of the technique is sufficient to permit the forensic scientist to establish the cause of death in “opiate sensitivity reactions” as well as those intoxications that involve high blood levels of morphine. The procedure has sufficient reliability for utilization in pharmacokinetic studies of morphine.
Eddy e t al. (1) described a quantitative gas-liquid chromatographic determination of morphine in 1961, one year following the demonstration by Lloyd et al. (2) that morphine analysis by gas-liquid chromatography was feasible. A plethora of gas chromatographic procedures for the detection of morphine has subsequently been introduced to the scientific literature. The earlier methods were directed to chromatographic separation of the free base (3-10) and explored the use of various column packings and treatment, but wereulimited by the nonlinear adsorption of the compound on the column. Investigators have derivatized morphine with a variety of reagents, achieving both superior chromatographic characteristics and enhanced sensitivity. Several authors utilized on-column derivatization techniques, forming morphine (1) N. B. Eddy, H. M. Fales, E. Haahti, P. F. Highet, E. C. Horning, E. L. May, and W. C. Wildman, United Nations Secretariat, ST/SOA/SER.K/114/ Corr. 1, Oct. 1961. (2) H. A. Lloyd, H. M. Fales, P. F. Highet, W. J. A. Vanden Heuvel, and W. C. Wildman. J. Amer. Cbem. Soc., 82, 3791 (1960). (3) L. Kazyak and E. C. Knoblock, Anal. Chem., 35, 1448 (1963). (4) K. D. Parker, C. R. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (5) E. Brochmann-Hanssen and T. Furuya, J. Pharm. Sci., 53, 1549 (1964). (6)J. L. Massingill, Jr., and J. E. Hodgkins, Anal. Cbem., 37, 952 (1965). (7) E. Brochmann-Hanssen and C. R. Fontan, J. Cbromatogr., 19, 296 (1965). (8) E. Brochmann-Hanssen and C. R. Fontan, J. Cbromatogr., 20, 394 (1965). (9) C. McMartin and H. V. Street, J. Cbromafogr., 22, 274 (1966). ( I O ) H. V. Street, J. Chromatogr., 29, 68 (1967).
acetate or propionate by injecting the appropriate anhydride immediately after the morphine injection (11-13); Anders and Mannering (11) in 1962 extended this technique to include trifluoroacetyl morphine and the trimethylsilyl ether. Most recent work, however, has centered on applications of the pre-chromatographic derivatization of morphine. The application of acetic anhydride to convert the free base to diacetylmorphine (heroin) was introduced in 1966 ( 1 4 ) and subsequently utilized by the authors (1.5). Trimethylsilyl ethers which utilize reagents such as hexamethyldisilazane (HMDS) (16-18), bis(trimethylsily1) acetamide (BSA) (19-20), and the trifluoro analog of BSA (21,22) have proved to be applicable to morphine analysis. Nalorphine, a structural analog of morphine, was observed to be an excellent internal standard for gas chromatographic determination of opiates. Street (10) first used nalorphine as an internal standard in the chromatography of free morphine base. Ikekawa e t al. (20) later demonstrated that nalorphine was excellent for derivatization applications in that the synthetic opiate served as a control for the derivatizing reactions as well as for the chromatographic techniques. With the exception of a few applications of argon ionization detectors (3, 1 6 ) , most gas chromatographic determinations of morphine have utilized flame ionization detectors (FID). Consequently, with morphine derivatives, the sensitivity of published methods is of such low magnitude that many procedures require 10 to 50 milliliters of urine or (11) M. W. Anders and G. J. Mannering, Anal. Chem., 34, 730 (1962). (12) S. J. Mule, Anal. Chem., 36, 1907 (1964). (13) H. W. Elliot, N. Nomof. K. Parker, M. L. Dewey, and E. L. Way, Clin. Pharmacol. Ther., 5 , 405 (1964). (14) E. Schmerzler, W. Yu. M. I. Hewitt, and I. J. Greenblatt, J. Pbarm. Sci., 55, 155 (1966). (15) J. E . Wallace, J. D. Biggs. and K. Blum. Clin. Cbim. Acta, 36, 85 (1972). (16) E. Brochmann-Hanssen and A. 6. Svendsen, J. Pharm. Sci., 51, 1095 (1962). (17) E. Brochmann-Hanssen and A. B. Svendsen, J. Pharm. Sc;., 52, 1134 (1963). (18) G. E. Martin and J. S. Swinehart. Anal. Cbem., 38, 1789 (1966). (19) F. Fish and W. D.C. Wilson, J. Cbromatogr., 40, 164 (1969). (20) N. Ikekawa. K. Takayama, E. Hosoya, and T. Oka, Anal. Biochem.. 28, 156 (1969). (21) G. R. Wilkinson and E. L. Way, Biochem. Pharmacol., 18, 1435 (1969). (22) H. E. Sine, N. P. Kubasik. and J. Waytash. Clin. Chem., 19, 340 (1973).
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10-100 ml of serum (blood). T h e method of Wilkinson and Way (21 ), using bis(trimethylsily1) trifluoroacetamide is a sensitive technique, applicable t o the quantitation of 25 nanograms of morphine in 1 ml of plasma, although t h e methods of Ikekawa et al. (20) and Wallace et al. (15) are also capable of determining nanogram amounts of morphine in biologic specimens. The introduction of electron capture detection (ECD) (23) has provided an extensively enhanced sensitivity for t h e detection of compounds having a high affinity for thermal electrons. Derivatization reactions using multi-fluorinated reagents have allowed gas chromatographic techniques utilizing ECD to detect sub-nanogram quantities of drugs in biologic specimens (24, 25). Selectivity becomes a limiting factor in such analytical techniques, for a number of substances normally present in biologic materials possess amino or hydroxyl functional groups capable of undergoing the same reactions as does the drug for which the analytical reaction is developed. Methods related t o electron affinity effects normally require elaborate and extensive extraction procedures, as illustrated by the procedures of Wilkinson and Way (21) and Ikekawa et al. (20) from which reported recoveries of morphine from biologic specimens approach 60%. The present report describes a gas chromatographic procedure applicable t o t h e determination of nanogram quantities of morphine in small volumes of biologic specimen, such as one milliliter of plasma. T h e method utilizes the electron affinity characteristics inherent in trifluoroacetylated morphine. Quantitation is based upon the relative peak heights of the trifluoroacetylated derivatives of morphine and nalorphine, the internal standard. The "cleanup" procedure is the combination of a previously reported gas chromatographic method (15) and a thin-layer chromatographic method (26). The technique proposed in this report provides a morphine recovery and purification that is most acceptable for electron capture methodology.
EXPERIMENTAL Apparatus. A Hewlett-Packard Model 5713A gas chromatograph equipped with a three-foot glass coiled column, 4-mm i.d. (3% OV-17 on Supelcon AW DMCS SO/lOO mesh, Supelco, Inc., Bellefonte, Pa.), and a 63Nielectron capture detector was utilized for gas chromatographic analysis. Chromatography was performed at column and detector temperatures of 220 "C and 300 "C, respectively, and a carrier (5% methane in argon) flow rate of 40 ml/ min. A Finnigan Model 3000 Gas Chromatograph Peak Identifier, equipped with a 1.5-foot column, 3% OV-1 on Gas Chrom Q, 100/ 120 mesh, was utilized for the mass spectrometric examination of the trifluoroacetylated derivatives. Reagents. Borate buffer, pH 8.9, consisted of a 0.050M boric acid-0.043M sodium borate solution. Phosphate buffer, pH 10.1 f 0.1, was prepared by adding additional amounts of disodium hydrogen phosphate as required to a 0.41M solution of same. An ethanolic solution of nalorphine (nalorphine HC1, Merck Sharp & Dohme Research Lab., West Point, Pa.) was diluted with ethyl acetate to provide a nalorphine concentration of 2 fig/ml. The ethylacetate (ACS Reagent Grade, Fisher Scientific) and trifluoroacetic anhydride (Aldrich Chemical Co.) were distilled prior to use. In addition to routine cleaning, all glassware utilized was exposed to an acid dichromate wash cycle. Procedure. Two milliliters of borate buffer and 1-2 ml of serum or plasma together with 25 ml of 10% isobutanol in chloroform were placed in a 50-ml glass stoppered tube and shaken vigorously for three minutes. The aqueous layer was aspirated off and the solvent centrifuged five minutes at 2000 rpm, after which it was decanted into a 25-ml glass stoppered graduate cylinder. The organic (23)J. E. Lovelock and S . R. Lipsky, J. Amer. Ch8m. Soc.,82,431 (1960). (24)M. Ervik, T. Walle, and H. Ehrsson, Acta fharm. Suecica, 7 , 625 (1970). (25)T. Walle and H. Ehrsson, Acta Pharm. Suecica, 8, 27 (1971). (26)J. E. Wallace, J. D. Biggs, J. H. Merritt, H. E. Hamilton, and K. Blum, J. Chromatogr., 71, 135 (1972). 2108
Table I. Gas Chromatographic Response to Trifluoroacetyl Morphine (Morphine/nalorphine peak height ratio)[
Morphine concn,
us/
0.125 0.250 0.50 0.75 1.0 1.5 2.0 2.5 3.0 5.0 7.5 10 15 20 25
Morphine/nalorphineQ. h
(morphine concn,
peak heightC ratio
ng/ul)
0.127 0.268 0.508 0.826 1.074 1.578 2.024 2.500 3.224 5.504 7.604 10.107 14.022 19.478 24.699
1.02 1.07 1.02 1.10 1.07 1.05 1.01 1.00 1.08 1.10 1.02 1.01 0.94 0.97 0.99
a All specimens contained 2.0 ng/pl nalorphine. b Mean relative standard deviation between duplicate determinations, 5.2%. c Typical absolute morphine peak heights: 0.25 ng/pl, 2.47-inch X atten 16; 2.5 ng/pl, 6.64-inch X atten 64.
layer at this point was washed twice with 5 ml of phosphate buffer (pH lO.l), and the recovered solvent volume recorded. Five ml of 0.5N HCI was added, and the cylinder shaken vigorously for three minutes. Four and a half ml of the acid extract were transferred to a glass stoppered tube, and the pH was adjusted to 8.7 f 0.2 by the addition of solid anhydrous potassium carbonate. The morphine was then extracted from the aqueous solution into 5 ml of ethyl acetate containing 10% by volume of isopropanol. Four and a half ml of the isopropanol-ethyl acetate solution along with 0.1 ml of the nalorphine solution were transferred to a 10-mm X 120-mm tube capable of being fitted with a Teflon-coated plastic screw cap, and evaporated to dryness under a gentle stream of filtered air at 50 "C. One-tenth ml each of ethyl acetate and trifluoroacetic anhydride was added, and the tube tightly capped and mixed vigorously on a vortex mixer. The tube was placed in a 50 OC water bath for 20 minutes, after which the contents were again evaporated to dryness as before. The specimens were reconstituted in 0.1 ml ethyl acetate, and 1-5 p1 aliquots used for chromatography. Quantitation was based upon the peak height ratios of the trifluoroacetylated derivatives of morphine and nalorphine.
RESULTS ECD Response. Reaction of morphine with trifluoroacetic anhydride under the conditions described above produces the di(trifluoroacety1ated) derivative as the principal product, as evidenced by mass spectra, although some of the mono-substituted material was detected. T h e vaporization temperature of the derivative was estimated to be approximately 192-4 "C. T h e solid cylindrical rod 63Ni ECD is very sensitive to t h e trifluoroacetylated morphine, the limit of detection being less than 0.1 nanogram. The derivatization is quantitative and the gas chromatograph response (peak heights) is linear with concentration over a wide range, as indicated in Table I. Reagent blanks typically exhibited minute peaks near t h e retention times of morphine and nalorphine corresponding t o a concentration of 0.03-0.04 ng/fiI; if higher blanks were observed, the ethyl acetate and TFA were redistilled. T h e relative retention time of trifluoroacetylated nalorphine to TFA-morphine is 1.35. Sensitivity for nalorphine
ANALYTICAL CHEMISTRY, VOL. 46, NO. 14, DECEMBER 1974
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Figure 2. Electron capture detector response to various concentrations of morphine extracted from plasma. Detector response is presented as the ratio of the peak heights obtained for the trifluoroacetylated morphine and nalorphine, the internal standard
-
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Figure 1. Representative chromatogram prepared from plasma containing a plasma concentration of 0.2 wg/ml morphine ( a ) . Nalorphine ( b ) , added as an internal standard to the solvent following extraction, at a final concentration of 2 ng/yl
is somewhat less than that for morphine. The morphine/ nalorphine peak height ratio was constant in individual studies (Table I), but over the course of our studies (which was a period of approximately two years) was observed to vary from 1.25 to 2.00, dependent upon factors such as age and prior conditioning of the column. This type of variability does not limit the application of the procedure for, in any gas chromatographic method, the analyst would be expected to include an internal standard with each group of biologic specimens that are to be analyzed. Plasma and Blood Determination. Determinations were performed on human plasma samples to which known quantities of morphine were added. The method results in clean chromatogams exhibiting good peak symmetry (Figure I), and a linear relation between plasma concentration and ECD response (Figure 2). The mean recovery of morphine, adjusted for volume recovery of solvents and 0.5N HCI, was 86%. The method offers possibilities of determining plasma (or serum) levels down to 25 ng/ml of morphine in a 2-ml specimen with acceptable precision, although concentrations of 5 ng/ml are detected by this procedure. Plasma specimens from adult open-heart surgery patients receiving morphine anesthesia (27) were analyzed. Of 72 specimens examined, 22 were less than 0.05 wg/ml and only 20 exceeded 0.20 Ig/ml. Volume of specimen analyzed was limited to a maximum of 2.0 ml of serum. Whole blood was obtained from adult mice that had received subcutaneous implantation of slow-release morphine/cellulose pellets in accordance with the technique of Gibson and Tingstad (28). A chromatogram from the analysis of a 0.5-ml specimen is shown in Figure 3. Interfering Pharmacologic Agents. A number of alkaline drugs were investigated for possible interference. Solutions containing 10 wg/ml of drug were subjected to the morphine trifluoroacetylation procedure and examined chromatographically. Darvon, demerol, amphetamine, meprobamate, quinidine, and acetaminaphen yielded no (27) C Petty, University of Texas Health Science Center, San Antonio. Texas, unpublished work, 1973 (28) R D Gibson and J E Tingstad, J Pharm S a ,59,426 (1970)
2
4
5
-IME
Figure 3. Chromatogram derived from 0.5 ml mouse whole blood specimen. ( a )is extracted morphine, ( b ) is the internal standard, nalorphine
peaks other than the solvent peak. Codeine and librium exhibited peaks with retention times relative to morphine of 1.4 and 2.3, respectively, but each was detected at levels that were significantly less sensitive than morphine. The retention times of the trifluoroacetylated derivatives of nalorphine and codeine are sufficiently similar (4.7 and 5.0 minutes, respectively) a t the chromatographic conditions of this report to prevent an effective separation of these compounds. The presence of codeine does not significantly impair the quantitative determination of morphine, however; codeine, having only one hydroxyl group available for trifluoroacetylation, offers an ECD sensitivity sufficiently low in relation to that for nalorphine that its presence does not significantly alter the nalorphine peak. The mean morphine/nalorphine (M/N) peak height ratio for solutions of ethyl acetate containing 1 ng/wl each of morphine and nalorphine and 10 ng/pl codeine was within 5% ( 5 determinations each) of the M/N ratio for samples containing an equivalent amount of morphine and nalorphine but no codeine. Procedural Evaluation. The use of chloroform, methylene chloride, and carbor. tetrachloride was examined for use in the extraction of morphine from plasma. Chloroform
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provided a 25% better recovery than that obtained with methylene chloride; carbon tetrachloride resulted in a very low yield of recoverable morphine. The use of isobutanol with chloroform resulted in a significantly enhanced extraction over that achieved by chloroform alone, and slight enhancement over the use of isopropanol and chloroform. Isopropanol and ethyl acetate were utilized as the solvent for the back-extraction to facilitate evaporation of the final biologic extract. For both the initial extraction and backextraction, 10% alcohol resulted in a higher recovery of morphine than could be achieved with 1%, and did not result in an equivalent increase in extraction of normal plasma constituents. Wallace et al. ( 2 6 ) have previously demonstrated that the phosphate buffer wash results in an insignificant loss of morphine, but eliminates considerable contamination from the biologic extracts. Most reactions requiring evaporative techniques generally require drying under a stream of nitrogen. Morphine and its trifluoracyl derivative are sufficiently resistant to oxidation that a stream of filtered air may be substituted for the nitrogen. Many procedures for the preparation of trifluoroacetylated derivatives are conducted in the presence of pyridine (Handbook of Silylation, Pierce Chemical Co., 1972, p 20). Pyridine is a most unsatisfactory solvent/catalyst for trifluoroacetylations of morphine, resulting in a yellow material which readily contaminates the detector. Yeh (29) used pyridine in the acetylation but not in the trifluoroacetylation of morphine. Within wide limits, the ratio of TFA and ethyl acetate (EtAc) is not critical. It was observed that decreased yields of trifluoroacetylated morphine were achieved if the amount of TFA relative to EtAc was very small (1-2%) or very large (>95%). No significant effect upon the yield was observed as the volume of reagents was varied from 0.1 to 1.0 ml, although the larger amounts of reactants required a longer evaporation time. Water vapor or aqueous contamination decompose both the TFA reagent and the trifluoroacetylated derivatives. It was observed that on humid days the derivatives were more stable providing ethyl acetate was added immediately after evaporation of the reactants. Instability of the trifluoroacetate derivatives in the dried residue was not observed on days that the humidity was less than 50%. Best results are obtained if the derivatives are chromatographed within a few hours of their preparation, although capped specimens of trifluoroacetylated morphine in ethyl acetate have been observed to maintin stability a t room temperature for periods of up to a week. If solutions of trifluoroacetylated morphine in ethyl acetate were stored at 4 "C or -20 "C, rapid decomposition occurred, apparently due to condensation of moisture within the enclosed atmosphere of the tube. A unique aspect of the reaction is that the optimal reaction temperature exceeds the boiling point of the reagent, trifluoroacetic anhydride, and consequently pressure builds up in the capped tubes during the incubation. Often imperfect seals developed resulting in decreased pressure within the tube and decreased morphine and nalorphine peak heights. Since both compounds were similarly responsive to the amount of TFA retained in the tube for derivatization, the morphine/nalorphine peak height ratios were consistent. In a study consisting of twelve standard solutions, each containing 5 ng/kl of morphine and nalorphine, the relative standard deviation of the absolute morphine peak height was f23.496, whereas the morphine/nalorphine peak height ratio varied only f4.0%.
The column used in this study requires pre-treatment prior to use to avoid undesirable adsorption of the trifluoroacetylated morphine. Commercial silylation agents are satisfactory, but it was preferable to inject, prior to utilization of the column, microgram quantities of the TFA morphine since that procedure does not necessitate removal of the column from the detector. Optimum reproducibility of the morphinelnalorphine peak height ratio was achieved by also pre-treating the column with TFA-nalorphine. The analysis for nanogram quantities of drugs in biologic materials necessitates rigid control of possible sources of contamination and variation in sensitivity. Reliable determination of morphine in amounts less than 100 nanograms requires acid-washing of glassware and the critical examination of each new lot of solvent and reagent. Certain lots of ethyl acetate and TFA were satisfactory as received, while others from the same commercial source required distillation prior to use. Disposable Pasteur pipets were used to direct the air stream in the evaporation steps and they required frequent replacement to avoid cross-contamination of specimens. Other sources of possible error are the presence of moisture, the lack of a sufficient seal of the reaction tube, and column adsorption due to inadequate treatment of the column. I t is important to note that each of these problems is diminished by the use of an adequate internal standard.
DISCUSSION The proposed method, by utilizing the electron capture detection of a polyfluorinated derivative and using a suitable internal standard, provides a sensitivity of morphine detection superior to existing gas chromatographic determinations of this compound. The procedure allows quantitative determinations of nanogram quantities of morphine in small specimens, i.e., 0.5-2.0 ml. Yeh (29) suggested tetraphenylethylene as the internal standard for use with the gas chromatographic examination of morphine derivatives. While this compound does possess a satisfactory retention time relative to morphine derivatives, it does not undergo the derivatization reaction and thus does not serve as a control for that portion of the procedure. Nalorphine, structurally identical to morphine in the vicinity of the hydroxyl groups, reacts similarly to morphine and thus serves as a control both for the derivatization reaction and for variations in the chromatographic technology. Flame ionization detectors, when utilizing trimethylsilyl or their trifluoro analog derivatives (16, 2 2 ) or trifluoroacetylated derivatives (11), offer a detector sensitivity limit of 2-10 nanograms of morphine. The electron capture detector (ECD), which was introduced over a decade ago (23), has a much superior sensitivity to electron absorbing species such as trifluoroacetylated derivatives. The earlier ECDs, which contained a thin foil of tritium or nickel-63 as the radioactive source, and used non-pulsed electronics, provided a very narrow range of linear response to concentration. The gas chromatographic system employed in the present study which has the G3Ni plated on the wall of the cylindrical cavity of the detector (30), significantly extended the linear response of electron capture detection to various concentrations of the TFA-morphine. The expanded range of linear response is primarily due to the mode of operation in that the electron population is sampled by frequency modulated pulses to produce a constant detector current. This particular type of ECD is also far more resistant to detector contamination and sample overloading, (30)R. J. Maggs, P. L. Jaynes, A. J. Davies, and J. E. Lovelock, Anal. Cbem.. 43, 1966 (1971).
(29) S. Y. Yeh. J. Pharm. Sci., 62, 1827 (1973).
2110
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problems which have hindered the successful application of previous ECDs to the analysis of biologic extracts.
David King, and Linda K. Goggin is deeply appreciated.
RECEIVEDfor review April 8, 1974. Accepted August 23,
ACKNOWLEDGMENT The technical assistance of John D. Biggs, Pamela Jones,
1974. This research was supported by Grant R01 DA00729 from the National Institute on Drug Abuse, NIH, Bethesda, Md.
Computer-Controlled Monitoring and Data Reduction for Multiple Ion-Selective Electrodes in a Flowing System J. J. Zipper,' Bernard Fleet,* and S. P. Perone3 Purdue University, Department of Chemistry, La fayette, Ind. 47907
A laboratory minicomputer system has been applied to the monitoring of ion-selective electrodes in a flowing stream. Up to 5 different electrodes could be monitored slmuitaneousiy. A standard addition analytical approach was implemented with a rigorous least squares fit to the data used to obtain electrode response slope and unknown analyte concentration for each electrode. The computer was programmed so that three different modes of data monitoring were possible: (1) Operator-controlled selection of data regions to be collected for subsequent analytical computations (STATIC program); (2) computer-controlled selection of data collection regions based on a pre-selected timedelay after each addition of standard (DUMB program); and ( 3 ) selection of data collection regions based on real-time computer identification of successive voltage plateaus In the potentiometer output during a series of standard additions (SMART program). Analog Instrumentation was developed which provlded a wide bandpass (to accommodate rapid multiplexing of electrodes), low drift, and high noise rejection. The digital instrumentation provided signal averaging, data sampling wlth f0.002 mV resolution, multiplexed sampling of 5 eiectrodeshec, and real-time digital display of signals from 5 electrodes. The entire system was evaluated by performlng a large number of standard addition experiments for fluoride analysis under optimized conditions. Analytical data could be obtained with a relative error of 0.4 to 3.3% and confidence Intervals varying from f0.4 to f1.6%. A comparison of the capabiilties of the different algorithms for data collection was made.
Ion-selective electrode (ISE) potentiometry is currently one of the more rapidly expanding techniques in analytical chemistry (1-3). A wide range ( 4 ) of electrodes responsive to cations, anions, enzyme-substrate systems ( 5 - 7 ) ,organic Present address, SPEX Industries, Box 798, Metuchen, N.J. Present address, Imperial College of Science and Technology, Department of Chemistry, South Kensington, London SW7, England. Author to whom reprint requests should be sent. (1) "Ion Selective Electrodes," R . A. Durst, Ed., Nat. Bur. Stand. (U.S.) Spec. Publ., 314 (1969). (2) R. P. Buck, Anal. Chem., 44, 270R (1972). (3) R. P. Buck, Anal. Chem., 46, 28R (1974). (4) R . A. Durst, Amer. Sci., 59, 353 (1971). (5) G. G. Guilbault. R. K. Smith, and J. G. Montaivo, Anal. Chem., 41, 600 (1969). (6) J. G. Montalvo, Anal. Chem., 41, 2093 (1969). (7) M. M. Fishman and H. F. Schiff, Anal. Chem., 44, 543R (1972)
ligands ( 8 ) ,and gases (9) are available, and new designs of electrodes continue to appear. One of the newly developing areas of application of these devices is in continuous monitoring (10-12). This approach appears to be particularly promising for applications to clinical analysis (13). The analytical utility of ion selective electrodes, however, is often impaired by two inherent fundamental limitations. First, the logarithmic relationship between electrode potential and primary ion activity limits the degree of accuracy attainable for a given measurement. The second limitation is that in many cases the electrode shows only moderate selectivity toward the primary ion of interest and the influence of interfering ions becomes highly significant. Selectivity limitations can in most cases be overcome by careful selection of the sample and the nature of chemical pretreatment. The accuracy attainable, on the other hand, is primarily dependent on the analytical technique used for the measurement. Direct potentiometry, although the most widely used approach, is the least accurate. Accurate matching of sample and standard is difficult and any electrode potential drift necessitates recalibration of the electrode a t a frequency determined by the degree of precision required and the rate of drift. The use of ion-selective electrodes as end-point sensors in titrimetric processes markedly improves the degree of precision attainable, although, with few exceptions (12), at the expense of considerable loss of convenience. The most convenient method for improving accuracy in direct potentiometry is by employing a standard addition approach (14, 1 5 ) . Either a single or multiple addition of the sought ion or a reagent which complexes the sought ion (standard substraction method) are possible. The graphical procedure of Gran can be employed (16, 1 7 ) , which involves a linearization of the standard addition equation and a multiple standard addition procedure. The use of computers to further enhance the accuracy (8)C. N. Wang, P. J. Kinlen, D. A. Schoeiier, and C. 0. Huber, Anal. Chem., 44, 1152 (1972). (9) "Newsletter," Orion Research, inc., Cambridge, Mass., Vol. V, No. 2, 197.1 n 7 '-'-> r
"
(10) T. S. Light, "Ion Selective Electrodes," R . A. Durst. Ed., Mat. Bur. Sfand. ( U S )Spec. Publ., 314 (1969). (11) B. Fleet and A. Y. W. Ho, "Ion Selective Electrodes," E. Pungor, Ed., Academia Kiado, Budapest, 1973. (12) 9.Fleet and A. Y. W. Ho, Anal. Chem., 46, 9 (1974). (13) G. A. Rechni!z. Amer. Lab., 6, 13 (1974). (14) "Newsletter, Orion Research, inc.. Cambridge, Mass., Vol. I, July, 1969, p 9. (15) /bid., September, 1969, p 25. (16) G. Gran. Analyst (London),77, 661 (1952). (17) A. Liberti and M. Mascini, Anal. Chem., 41, 676 (1969).
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