Anal. Chem. 1980, 52, 2383-2387 (9) Schenck, P. K.; Mallard, W. G.; Travis, J. C.; Smyth, K. C. J . Chem. Phys. 1978, 69, 5147-5150. (10) Travis, J. C.; Schenck, P. K.; Turk, G. C.; Mallard, W. G. Anal. Chem. 1979, 57, 1516-1520. (11) Green, R. B.; Havrilla, G. J.; Trask, T. 0. A w l . Spectrosc. 1980, 3 4 , 561-569. (12) Smith, 8. W.; Parsons, M. L. J. Chem. Educ. 1973, 50, 679-681. (13) Havrilla, G. J.; Green, R. B., submitted for publlcation in Anal. Chem.
RECEIVED for review June 30, 1980. Accepted September 18,
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1980. This research was supported by the National Science
Foundation under Grant No' CHE-79-18626*This work was presented in part a t the 179th National Meeting of the American Chemical Society, Houston, T X , March 1980. This paper was taken in part from the dissertation written by G. J. Havrilla in partial fulfillment of degree requirements for a Doctor of Philosophy in Chemistry from West Virginia University, Morgantown, WV.
Disposable Potentiometric Ammonia Gas Sensors for Estimation of Ammonia in Blood M. E. Meyerhoff" and R. H. Robins Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48 109
A simple, rapid, and Inexpensive method for estimating the ammonia content of various blood samples is presented. The method utilizes a newly developed ammonium selective membrane electrode-based potentiometric ammonia gas sensor in conjunction with a multiple standard addition procedure. The assay requires 250 pL or less of sample and is shown to yield good accuracy (relative errors, -7.0 to +14%) and precision (relative standard deviation, 2.5-12.0 YO) in the normal ammonia concentration range. Results obtained for pooled human plasma and serum samples correlate well with a current manual enzymatic procedure. Additional Information concerning the design and analytical function of the new disposable gas sensor is also presented.
The application of membrane electrodes and sensors for the direct determination of discrete ions and gases in physiological samples has grown rapidly in recent years (1-4). Potentiometric sensors offer several significant advantages for such measurements, including, simple instrumentation requirements, minimal additional reagents, low cost, and freedom from sample turbidity and color problems which often interfere with the more traditional photometric assays. We have recently introduced a new type of potentiometric ammonia gas sensor ( 5 ) . In this paper we present a method for using this disposable sensor for the rapid estimation of ammonia in serum, plasma, and whole blood samples. (Throughout this paper, in order to be consistent with previous clinical chemistry literature, the terms ammonia or ammonia in blood refer to the total concentration of ammonia gas plus ammonium ions present, i.e., total ammonia nitrogen concentration.) T h e measurement of ammonia in blood is an important diagnostic test for several disease states including hepatic coma and the fatal childhood disorder, Reye's Syndrome (6). Recent outbreaks of Reye's Syndrome have demonstrated the need for a rapid, simple, and reliable method to detect ammonia in blood; perhaps one which could readily be performed in a small laboratory or physician's office. Manual determinations of ammonia can be made by a number of methods, including ion exchange (7), isothermal diffusion (8),and enzymatic assay (9, 10). The ion-exchange and isothermal procedures are slow and require many sample manipulation 0003-2700/80/0352-2383$01 .OO/O
steps which can decrease the accuracy of these techniques. The enzymatic assay employs glutamate dehydrogenase (GLDH, EC no. 1.4.1.2) to catalyze the reaction
NH4+ + NADH
+ a-ketoglutarate
-
glutamate
+ NAD
(1)
The decrease in absorbance a t 340 nm, due to the disappearance of reduced nicotinamide adenine dinucleotide (NADH) from the assay mixture, is proportional to the ammonia present. T h e manual method suffers from poor precision and accuracy in the normal ammonia concentration range ( I I ) , and other drawbacks, including interferences from competing enzymatic reactions, generation of ammonia during the reaction, and interferences due to GLDH inhibitors which may be present in the sample (i.e., drugs) (12). Normal values of ammonia found in blood vary considerably depending on the method used. For most blood specimens, normal values ranging from 20 to 80 kmol/L have been reported (13) with whole blood levels usually slightly higher than serum or plasma values. Commercially available ammonia-selective gas sensors, utilizing p H glass membranes as internal sensing elements, have previously been used for blood ammonia determinations (12,14-16). Use of these sensors requires operation a t a pH >10.3 so that all ammonia in the blood sample is present as free dissolved gas. Under these alkaline conditions, the labile amide groups of the amino acids glutamine and asparagine may hydrolyze to give false elevated ammonia values and drifting electrode potentials (12-14). Prior perchloric acid precipitation of proteins has been employed (12) to reduce these problems, but increased sample handling and adjustment of the sample to pH 11.0 is still required. In addition, the size of the commerical ammonia sensors has necessitated the use of the rather large volumes of blood samples (2-3 mL). Here we describe a simple, multiple standard addition procedure to directly estimate the concentration of ammonia in serum, plasma, or whole blood. The method employs a newly developed polymer membrane electrode-based ammonia-selective gas sensor (5) which has improved detection limits over existing commercial sensors. Measurements take place under mild buffer conditions, p H 8.5, and, therefore, the possibility of hydrolysis reactions which liberate additional ammonia is greatly reduced. Furthermore, the amount of sample required for the assay is 250 FL or less. Potentiometric 0 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
F - s m m i
Figure 1. Schematic diagram of disposable ammonia sensor: (a)plastic pipette tips, (b) coaxial cable, (c)Ag/AgCI electrodes, (d) parafilm (Iring, (e) internal electrolyte (0.01 moVL ",GI), (f) Tygon tubing, (g)internal buffer, (h) PVC-nonactin membrane, (i) plastic O-ring,0)gas permeable
membrane. measurements made on pooled serum and plasma samples correlate well with a standard manual enzymatic method. In addition, we provide further fundamental information concerning the design and optimization of the new disposable ammonia sensor.
EXPERIMETNAL SECTION Apparatus. Potentiometric measurements were made by wing a Corning Model-12 pH meter in conjunction with a HeathSchlumberger Model 204 recorder. All calibrations and sample measurements were made in a 10-mL cell, thermostated at 25 f 0.1 "C. Spectrophotometric enzymatic assays of ammonia in plasma and serum samples were performed by using a Cary 219 spectrophotometer. Computer calculations and programs were run on a Commodore PET minicomputer (16K), programmed according to the methods outlined below. Microliter quantities of NH4C1standards were added to the assay solutions by using variable Finnpipettes (Variable Volumetrics Inc.). Reagents. An enzymatic reagent kit for the determination of ammonia in plasma was obtained from the Sigma Chemical Co. and used as recommended for low-level ammonia measurements (Le., 0.3 mL sample rather than 0.2 mL). All other chemicals were reagent grade. Buffers and other aqueous solutions were prepared with distilled-deionized ammonia-free water. The following working buffers were prepared fresh weekly: 0.1 mol/L tris(hydroxymethy1)aminomethane-hydrogen chloride (Tris-HC1) pH 8.5 (assay buffer) and 0.01 mol/L Tris-HC1, pH 7.5, containing 0.2 mol/L glucose (sensor's internal buffer). (The concentration of buffers in moles per liter refers to ionic strength.) Two lots of lyophilized commercially available normal control serums were obtained from Fisher Scientific (Serachem, Lot No. 2905-602) and Scientific Products (Dade, Monotrol I, Lot No. LTD-158) and reconstituted with distilled water. Preparation of Disposable Ammonia Selective Gas Sensors. Several minor modifications have been made in the design of these new polymer membrane based ammonia electrodes since our original study (5). Figure 1 schematically represents this new sensor and the novel fashion in which the sensing body (disposable) slips snuggly onto the silver/silver chloride reference electrodes (nondisposable). All components of the sensor are readily prepared in the laboratory. The sensor body consists of three plastic Finnpipette tips carefully cut to proper dimensions. The entire sensing portion of the electrode costs pennies to make. For this study, the size of the electrode has been increased slightly to a 5 mm tip diameter. This facilitates the preparation of the electrode and greatly reduces noise problems encountered with the previous 3 mm size (5). Additional noise reduction was obtained by using thinner PVC-nonactin polymer membranes (0.10 mm thick). This minimizes electrode resistances so that no special high input impedance devices are required to make
potential measurements. The gas permeable membrane material used here was exclusively poly(tetrafluoroethy1ene) (0.2 pm pore size, W. L. Gore Inc.). The geometry of the sensing tip was also much more carefully controlled in this work than it was in our initial study. The inner ammonium sensing polymer membrane tip (Tygon tubing (f) in Figure 1) fits rather tightly into the lower portion of the outer pipet tip so that minimal space is left for sample gas diffusion into the bulk solution. Too tight a fit, however, may eliminate electrolyte contact with the reservoir of buffer and lead to erratic electrode behavior or open circuits. Procedures for Sensor Evaluation and Blood Ammonia Estimations. The ammonia gas sensors made in our laboratory were initially evaluated for electrode response by obtaining calibration curve data in a 0.1 mol/L Tris-HC1, pH 8.5, working buffer. This was accomplished by making additions of standard NH4C1solutions to the working buffer. Once it was determined that the sensors exhibited acceptable response properties, they were used for subsequent blood measurements. Nonidentifiable pooled plasma, serum, and whole blood samples were obtained from the University of Michigan Hospital. Plasma samples obtained were stored at -30 'C until assayed (several weeks). Serum samples were assayed on the same day that they were drawn, although they were stored, refrigerated, for several hours before the measurements were actually made. Fresh, whole blood samples were immediately frozen and stored for 1day until assayed. Potentiometric data were obtained as follows: The sensor was always stored in fresh well-stirred working buffer between measurements. For assays of blood samples and aqueous NH&l standards, 750 p L of working buffer was pipetted into a small thermostated cell and stirred with a small Teflon stirring bar. The sensor was placed into the solution and allowed to reach rapid equilibrium potential (base line-no ammonia). Fifty microliters of sample was then added and the potential change observed. If the sample appeared to have relatively low ammonia content (0.9998). Increasing the ionic strength and buffer capacity of the internal buffer to 0.1 mol/L Tris-HC1 with 0.1 mol/L glucose added, effectively extends the linear portion of the curve to >1 X mol/L ammonia but a t the expense of slightly poorer detection at lower levels. This is because the nonactin
' J Flgure 3. Typical strip chart recording showing the response of the new ammonia-selectbesensor to increasing concenVations of ammonia mol/L in 0.1 mollL Tris-HCI, pH 8.50. Also shown is recovery time in fresh buffer. (Trace shown is for different day than plotted in Figure 2; for linear range, E = -50.5 X -I-117.1.)
t
I
l
1
1
1 3
5
7
A g e , ldayl
Figure 4. Time study of absolute potentials observed with new amrwnia electrode at varying ammonia concentrations, mol/L. membrane has a slight response to the increased concentration of protonated Tris. For optimum detection limits in the blood assays, we used the lower ionic strength internal buffer for most of our measurements. Following the careful preparation steps outlined in the Experimental Section, the new gas sensors display remarkably short response times to reach equilibrium potentials. Figure 3 shows a typical strip chart recording trace of the electrode response upon additions of NH4C1to the working Tris-HC1 buffer. Even at the low end of the calibration curve, response times are rather short,
