On-line gas dialyzer for automated enzymatic analysis with

luent reagents, length of dialysis tubing, etc. are described. Potentiometric gas sensors, particularly the ammonia gas sensor, have become useful ana...
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Anal. Chem. 1983, 55, 359-364

LITERATURE CITED

(7) Proskurnin, M.; Frumkin, A. N. Trans. Faraday SOC. 1935, 3 1 . iin-iic ," (8) Mellk Gaikazyan, V. I. Zh. Flz. Khim. 1952, 2 6 , 560-580. (9) de Levie, R.; Pospisil, L. J . Nectroanal. Chem. 1969, 22, 277-290. (10) de Levie, R. Anal. Chem. 1980, 52, 1535-1537. I

(1) Seelig, P. F; de Levie, R. Anal. Chem. 1980, 52, 1506-1511. (2) Conway, B. E.; Kozlowaka, H. A,; Sharp, W. R. A.; Griddle, E. E. Anal. Chem. 1973, 45, 1331-1336. (3) Matsuda, H. Z . Elektrochem. 1958, 62, 977-989. (4\ de Levie. R.: Husovskv. A. A. J . Electroanal. Chem. 1969. 20. 181-193. (5) Smith, D. E. Electroanal. Chem. 1966, 1 , 1-155. (6) de Levie, R; Thomas, J. W.; Abbey, K. M. J . Electroanal. Chen?. 1975, 62. 111-125.

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359

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RECEIVED for review August 27, 1982. Accepted November l2,1982* This work was by the Air Force Office of Scientific Research under Grant AFOSR 80-0262.

On-Line Gas Dialyzer for Automated Enzymatic Analysis with Potentiometric Ammonia Detection Y. M. Fraticelli' and M. E. Meyerhoff" Department of Chemistry, Universi@ of Michigan, Ann Arbor, Michigan 48109

An automated enrymatlc assay system Incorporating ammonia electrode detection in conjunction with a novel on-line predlalysls unlt is descrlbed. The predialysis unlt nondestructlvely and efficiently removes Interfered normal and abnormally high backgraiund levels of ammonla nitrogen from phydologlcal samples prior to the enzymatic step. The cllnical applicatlon of the system Is demonstrated vla the dlrect measurement of L-asparagine In plasma samples through the use of the enzyme L-asparaginase In the soluble form. Peak potentials observed are logarithmically related to the sample substrate concentration. At least 30 sampleslh can be assayed with excellent precision for asparagine concentrations Iio-' mol/L. Fundamental lnvestigatlons concernlng the optlmiratlon of the system varlables such as flow rates, dlluent reagents, length off dialysls tublng, etc. are described.

Potentiometric gas sensors, particularly the ammonia gas sensor, have become useful analytical detectors for the determination of amino acids and other biomolecules (1-11). In these assay systems, various biocatalysts (e.g., enzymes, bacterial cells or tissue slices) are either immobilized on the surface of the gas sensoie's membrane (1-8) or, as in a flowthrough arrangement, the biocatalysts are used in soluble form or are immobilized in a tubular reactor (9-11). These biocatalysts serve to selectively convert the biomolecule to ammonia (so called "deaminase" enzymes). Bioanalytical systems utilizing flow-through arrangements are more appealing since there is no need to compromise the optimum conditions needed for biocatalytic activity and maximum sensor response. In the development olf sensitive and accurate assays for amino acids and other metabolites in physiological fluids through the use of ammonia liberating catalysts, interference from background levels of ammonia nitrogen (NH,-N) is encountered. Normal NH3-N in blood samples is about the same or slightly lower than norinial amino acid and metabolite levels (12,13). However, if there is any delay in the analysis of the sample, these background NH3-N levels will greatly increase due to the hydrolysis of blood components into ammonia (14). This increase in NH3-N can interfere with the accuracy of the 'Present address: Polaroid Corp., 750 M a i n St., Cambridge, M A

02139. 0003-270O/83/0355-0359$0 1.50/0

assay. Consequently, physiological samples have to be pretreated by various tedious and time-consuming methods such as cation exchange (11,15) or alkalinization followed by heat evaporation (16,17). These pretreatment steps are not needed to accurately measure urea in blood samples when using the enzyme urease and an ammonia gas sensor (9, 18). This is because the physiological range of urea is to lo-' mol/L which is a t least 1 order of magnitude greater than normal mol/L). However, when NH3-N levels to 8 X measuring amino acids or metabolites in physiological samples, which are normally present in much lower concentrations than urea, the background NH3-N levels do pose a problem. For example, Mascini and Palleschi (11)recently reported on the development of an automated system for the determination of creatinine in blood and urine samples utilizing an immobilized creatininase reactor and ammonia gas sensor. For accurate creatinine values, the blood NH3-N had to be determined first and then subtracted from the creatinine plus ammonia response. The NH3-N in the urine samples had to be removed by a cation exchange method prior to analysis. In this report, we describe an automated electrode-based ammonia-liberating enzymatic assay system which can accurately and rapidly measure the amino acid L-asparagine in blood samples. The assay is virtually free from interference by background NH3-N in the sample. The system utilizes a previously described automated polymer membrane based ammonia detection device (19) in conjunction with enzymatic reagents in solution which selectively liberate ammonia from various substrates. The problem of NH3-N levels normally present in the samples is overcome by incorporating a unique on-line gas dialyzing unit which nondestructively and efficiently removes the NH3-N background as the sample is introduced into the system. consequently, the amount of NH3-N that is liberated from the enzymatic reaction is directly proportional to the substrate concentration present in the sample. Emphasis has been placed on optimizing the system for the determination of the amino acid L-asparagine. However, the system described can be readily modified for assaying other amino acids or metabolites of clinical importance when the appropriate biocatalysts, either in solution or immobilized in a tubular reactor, are used. The system can accurately determine L-asparagine levels below mol/L and at least 30 samples/ h can be assayed with high precision in the normal blood plasma asparagine range. Under present conditions, even abnormally elevated levels of background NH3-N in 0 1983 American Chemical Society

360

ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983

n SAMPLER

,

ar

+

+ + +

NH3

NH3 NH3

t I

NH3

r H 3 N,H3

- - - -

V

I

N73 V

'

NH3

,

v

'

NH NH3

,

*SAMPLE-

G

- - -- - - -

Figure 1. Schematic diagram of automated electrode-based enzymatic assay system: (REC) recorder; (pH)pH-mV meter; (r) saturated calomel

reference electrode; (e) electrolyte solution; (Sb) salt bridge; (fte) flow-through polymer-membrane electrode unit; (9) electrical ground; (m) gas-permeable membrane; (dc) dialysis chamber; (ps) pulse suppressor; (d) debubbler; (mc) seven-turn mixing coil; (RC) reaction coil; (tc) Teflon dialysis coil; (ar) acid reservoir; (w) waste, Insert illustrates expanded view of dialysls unit and chemical processes which take place within. Numbers represent flow rates in mL/min.

plasma samples will not significantly interfere with the accuracy of the assay.

EXPERIMENTAL SECTION Apparatus. potentiometric measurements were made with an Altex Select-Ion 2000 ion analyzer (Scientific Products, Romulus, MI) and recorded on a Houston Instruments (Austin, TX) Omniscribe strip-chart recorder. The automated gas-sensing arrangement consisting of a gas dialyzer chamber, a flowing internal electrolyte technique, and a tubular nonactin-PVC membrane electrode was assembled as previously described (19) except that the manifold was modified to handle enzymatic reactions and an on-line predialysis unit was incorporated into the system. The predialysis unit consists of a 4.2 m length of Teflon gaspermeable tubing (poly(tetrafluoroethylene), 1mm i.d.) obtained from W. L. Gore and Associates (Elkton, MD) which was coiled and placed into a 1-L acid reservoir. All measurements were made a t room temperature. Reagents. All chemicals used were of reagent grade. Standard solutions and buffers were prepared with distilled-deionized water. Buffers and solutions studied as potential diluents, pH adjustors, and dialyzing medium included the following: tris(hydroxymethy1)aminomethanehydrochloride (Tris-HC1,pH 8.6,0.05 mol/L), (cyclohexy1amino)propanesulfonicacid (CAPS, pH 10, 0.03 and 0.10 mol/L), citrate buffer (pH 4.0, 0.10 mol/L), HCl (0.10 mol/L), and NaOH (0.00036,0.001,0.03,0.50,0.25,and 0.20 mol/L) (buffer concentrations refer to total ionic strength). Tris-HC1 (pH 7.5,O.Ol mol/L) was used in the flowing internal electrolyte stream. The amino acids, L-asparagine and L-glutamine, were obtained from Sigma Chemical Co. (St. Louis, MO). The enzyme L-asparaginase,Grade VIII, from E. coli (E.C. 3.5.1.1), was also a product of Sigma Chemical Co. A stock solution of 1 mg/mL (192 U/mg) was prepared in Tris-HC1 (pH 8.6, 0.05 mol/L) placed in a dialysis tube (Union Carbide Corp., Chicago, IL) and dialyzed for 48 h at 4 OC prior to use. Any unused portion

of the stock was kept under constant dialysis in order to prevent NH3 background buildup which occurred with time. Evaluation of Predialysis Unit. Figure 1 illustrates a schematic diagram of the flow arrangement used to evaluate the efficiency of our on-line predialysis unit. The various flow rates, sample to diluent ratios, various pH ranges, etc. which were studied in order to achieve optimum NH3background removal from the sample will be discussed in more detail in the Results and Discussion Section. Initially, the predialysis unit is removed. Aqueous NHICl standards ranging between and mol/L are then introduced into the system and peak potentials recorded. The predialysis unit is then reincorporated into the system. Fresh NHICl standards are introduced, mixed with NaOH 0.03 mol/L to adjust the pH of the sample stream to favor the formation of NH3 gas, and passed through the predialysis unit. In an analogous fashion, serum samples ranging within normal to abnormal levels of NH3-N were utilized in order to evaluate the predialysis unit in its efficiency to remove NH3-N from real samples. The various levels of NH3-N in the serum samples were attained by spiking 3-mL aliquots of pooled serum obtained from the University of Michigan Hospital (Ann Arbor, MI) with microliter amounts of and lo-' mol/L NH&l standards. Procedure for Blood Plasma Asparagine Determination. A calibration plot of aqueous L-asparagine standards ranging between and mol/L was recorded prior to introducing the plasma samples using the enzyme L-asparaginasein the diluent buffer stream of the system. The sampling rate was 30 samples/h with a sample to wash ratio of 1:2. Plasma samples were obtained from healthy members of our research group. Blood was drawn into heparinized evacuated tubes in our own laboratory and placed immediately on ice. The blood plasma was obtained by centrifuging the blood samples for 5 min under refrigerated conditions. All of these samples were assayed within 30 min.

ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983

Plasma NH3-Nlevels were assayed by removing the predialysis unit and utilizing enzyme-free Tris-HC1, pH 8.6, buffer in the biocatalyst stream prior to assaying for L-asparagine. Unknown blood plasma NH3-N and L-asparagine levels were determined from the prior aqueous calibration standards using a linear “least-squares fit” of the data. Procedure for Blood Plasma Recovery Studies. To each of 2-mL aliquots of pooled blood plasma, microliter amounts of mol/L, were added, mixed, and introduced fresh L-asparagine, into the automated system. The L-asparagine content in the spiked samples was determiied from a ”least-squares fit” obtained from an aqueous L-asparagine calibration curve.

RESULTS AND DISCUSSION The objective of this study was to develop a simpler and more convenient amino acid and metabolite assay system based on deaminating enzymes, a potentiometric ammonia gas detector, and a sample pretreatment step all in one automated continuous-flow unit. Figure 1illustrates this device. The key component of this automated system is the on-line predialysis unit. The insert in Figure 1 shows an expanded view of the predialysis unit and the chemical processes which take place within. Upon passage of the sample through this unit, the background NH13-N present will diffuse through the gas-permeable walls of the Teflon tubing (tc) and is trapped as ammonium ions by the acid reservoir (ar). Amino acids and other metabolites present in the sample cannot permeate the walls of the Teflon tubing, and therefore, they remain at their original levels in the sample. The extent of NH3-N removal can be seen by comparing the peak potentials obtained when the sample is passed through the system without the use of the predialysis unit and when the unit is used. In the design of the final system illustrated in Figure 1, numerous fundamental studies were undertaken in order to achieve optimum sample NH3-N removal, as well as maximum sensitivity for the amino acid assay. Results and discussion of these studies are as follows: Choice of Sample Diluents and Dilution Ratios. In the sample pretreatment step, it is important that the NH3-N present in the sample is as much in the form of free gas as possible. From the pK, of the ammonium-ammonia equilibrium, 9.3, the ammonia gas species will predominate a t pH values of 10 or above. Considering that the system being developed is for the assay of amino acids in physiological fluids, care was taken to keep the p H of the sample diluent mixture below 11 in ordeir to prevent the hydrolysis of labile amines present (11,19,20). Sodium hydroxide (0.00036,0.001, and 0.03 mol/L) and the buffer CAPS (pH 10, 0.03 and 0.10 mol/L) were studied as potential diluents. These solutions were mixed in a 1:l and 1:2 sample to diluent ratio with pooled serum samples whose initial pH was 8.45. The final pH of the serum-diluent mixture was measured manually. The buffer CAPS was not able to completely adjust the pH of the serum sample to 10 even when an ionic strength of 0.10 mol/L was used. Higher ionic strengths were not attempted since higher buffering capacity could interfere with readjusting the sample stream to a lower pH with a secondary buffer-diluent for optimum enzymatic conditions. Sodium hydroxide, 0.03 mol/L, in a 1:2 dilution ratio was able to adjust the serum pH to 10.6, a value at which the NH3-N is 95970 in the free ammonia form and still mild enough to prevent hydrolysis of other components in the serum. In addition, NaOH, not being a buffer, would not interfere with the secondary buffer diluent’s capacity to readjusk the sample stream’s p H to favor the enzyme. The dilution ratio of sample to diluent used throughout was 1:2 in order to minimize the viscosity of the serum or plasma sample. Due to the short iresidence time the sample has in the predialysis coil (ca. 82 s), high sample viscosity would interfere with the diffusion of the free ammonia gas and impair

361

the efficiency of the NH3-N removal. Larger dilution ratios would be favored but at the expense of decreasing the system’s sensitivity since the sample is further diluted as it continues through the network. The diluent buffer system used for the enzymatic step of the assay was Tris-HC1, pH 8.6, 0.05 mol/L. This buffer is the one recommended to be used with the enzyme Lasparaginase (21). An ionic strength of 0.05 mol/L was used in order to ensure that the pH of the blood plasma and serum samples were adjusted to 8.6. The total sample dilution in the entire system is approximately 1:7. In order to overcome loss in sensitivity at pH 8.6, due to our high dilution ratio, we again adjusted the sample stream to pH 11.0 by introducing NaOH, 0.25 mol/L. Consequently, at this pH, the enzymatic reaction is also quenched. Under these conditions very efficient ammonia removal was achieved and enzymatic conditions and electrode response properties were optimal. Acid Reservoir Reagent. As the sample travels through the predialysis unit, the ammonia that permeates through the walls of the Teflon coil is trapped as ammonium ions by the solution that makes up the acid reservoir. A sodium citrate buffer (pH 4.0, 0.10 mol/L) and HCl (0.10 mol/L) were studied as potential acid reservoir solutions. Hydrochloric acid (0.10 mol/L) proved to be the more efficient dialyzing medium of the two due to the larger pH difference between the sample stream inside the coil and the bulk solution. During the course of all the studies, the acid reservoir container was open to the atmosphere and the same acid solution was used for a t least 3 days without any loss in its capacity to trap ammonia. Air Segmentation vs. Nonsegmentation through Predialysis Unit. Initially, the sample and NaOH diluent were mixed without the use of air segmentation. The only air segment present was that produced by the sampler as the probe shifted from the wash reservoir to the sample cup. As the sample segment passed through the predialysis unit, this small air segment was lost by diffusipg t,hrough the gaspermeable Teflon coil. The consequence of this air segment loss was large sample dispersion, even though the dialyzed sample stream was air segmented when mixed with the secondary buffer diluent stream. Air segmentation minimized sample dispersion and thorough sample mixing was ensured. A large portion of each air segment was lost during dialysis but what remained was sufficient to keep the sample stream segmented. In addition, the remaining air segments were small enough not to interfere with the reintroduction of air segments when the sample stream mixed with the enzyme/buffer stream. Length of Dialysis Coil. Two different Teflon tubing lengths were studied. The first dialysis unit tested utilized a 2.1-m length of Teflon tubing. This coil length proved to be quite efficient in removing NH3 from aqueous samples (>95%) ranging between 10-5and mol/L. However, when real samples were assayed (Le., serum), efficiency of NH3-N removal decreased to about 80%. The high viscosity of serum or plasma samples will interfere with the diffusion rate of NH3 from the sample. The length of the dialysis tubing was doubled (4.2 m) in order to double the residence time of the sample in the dialysis unit. Efficiency of NH3-N removal from the serum and plasma samples was >90% with the double length coil. Flow Rates, Sampling Rates, and Sample to Wash Ratio. In our previous report (19) high precision, accuracy, and low detection limits were obtained in the automated NH3-N assay system when a sampling rate of 30 samples/h and a sample to wash ratio of 1:2 were used. Since this automated enzyme based assay system utilizes the same detection scheme as the NH, system, the same sampling rate

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ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983

and sample to wash ratio were used as well as the flow rates for the internal electrolyte and sample streams through the gas dialyzer electrode assembly. For a final flow rate of 3.90 mL/min in the sample stream after debubbling, the sample stream flow rate (excluding air segment flow rates) should be at least 20% higher, or 4.70 mL/min in order to avoid the pull of air bubbles through the gas dialyzing chamber. Minimal sample consumption, the initial 1:2 dilution, length of time the sample will spend in the predialysis unit, and the approximate 1:ladditional dilution with the biocatalyst diluent stream were parameters that were taken into consideration when flow rate optimization was studied. The optimum flow rates attained for this system are shown in Figure 1. Determination of L-Asparagine. In an attempt to measure L-asparagine in aqueous and blood plasma samples, we incorporated a 12.2-m glass coil (2.4 mm i.d.) into the network along with a special mixing coil (Technicon P / N 116-0133-01)which allowed the introduction of an additional reagent stream. The 12.2-m coil was used as a reaction chamber (RC) which allowed the sample and the biocatalyst reagent to mix and react for a fixed time period (approximately 9.0 min). Upon completion of this reaction time period, the reaction quenching and pH adjusting reagent was introduced into the sample stream via the special mixing coil. The amount of enzyme activity/unit volume to completely hydrolyze the amino acid to its product and NH3 L-asparagine

+ H20

L-asparaginase

L-asparate

1000

BLOOD PLASMA

I1 O ' !l;

Flgure 2. Tracing of a typical stripchart recording of ammonia-N levels in nine fresh plasma samples: (A) prior to addition of dialysis unit; (B) after passing through dialysis unit. Calibrants are ammonium chloride standards in pmol/L concentrations. Conditions: sample to wash, 1:2; sampling rate, 30/h; inltial dilution with 0.03 mol/L NaOH, 1:2; second dilution with 0.05 moVL Trls-HCI, pH 8.6 is approximately 1:1.4; electrolyte, 0.01 mol/L Tris-HCi, pH 7.5; final pH adjustor, 0.25 mol/L NaOH. Drop in base line potential (10 mV) is due to the dialysis of trace levels of ammonia-N present In reagents. On an absolute potential scale, the remaining ammonia-N levels in plasma samples after dialysis are 5 1 pmol/L.

+ NH,

was calculated on the basis of (1)how much sample is aspirated, (2) the concentration of the upper most normal limit of the amino acid assayed, (3) the enzyme's activity, (4) the flow rate at which the enzyme is introduced into the system, and (5) the length of the enzymatic reaction time. Once the amount of enzyme activity needed is calculated, a 10-fold excess is utilized to ensure total conversion of the substrate. The determined enzyme concentration was tested under the established working conditions shown in Figure 1. The peak heights recorded from the enzymatic hydrolysis of Lasparagine were compared to those obtained from NH4C1 standards under the same conditions. The peak heights (hE's) from the NH4C1 standards were slightly higher than the Lasparagine peak heights indicating incomplete substrate conversion within the allotted reaction time period. However, the peak heights for both assays were reproducible to *1.3 mV or less (standard deviation of four measurements) between the and mol/L range. This precision corresponds to a relative error of I f 5 % for measurements made in this region. The percent conversion ranged from 82% at mol/L to 72% a t mol/L L-asparagine. This incomplete substrate conversion could have been influenced by such factors as dilution ratios, which lowered the substrate conmol/L (22))and centration to below its K , value (6 X working a t suboptimum temperature (25 O C vs. 37 "C). However, the response slope obtained for the L-asparagine standards in the to mol/L range was 58.09 mV/ decade and linear throughout the concentration range (correlation coefficient 0,9999). Nine fresh nonfasting blood plasma samples were obtained and assayed by this automated enzyme system. The background NH3-N of the sample was determined utilizing this system (see Experimental Section), without using the predialyzer, prior to assaying for L-asparagine. Figure 2 shows a tracing of the potentiometric recording of the background NH3-N levels of the fresh plasma samples (A), along with a recording illustrating the efficiency of the on-line predialysis unit in removing this interfering NH3-N background (B). In Figure 2, the resultant NH3-N levels in the samples (B), compared to the absolute peak potentials obtained from the

1000

I

CALIB. PLASMA

L-ASPARAGINE

Flgure 3. Tracing of a typical strip-chart recording of plasma sample measurements for asparagine. The calibrants are L-asparagine standards in pmoi/L concentrations. Conditions are the same as those given in Figure 2 except that L-asparaginase (2 X lo-* U/mL) is added to the secondary diluent buffer solution.

Table I. Summary of Ammonia-N and L-Asparagine Values Found in Normal Blood Plasma Samples sample 1 2 3 4 5 6 7 8 9

amt found, +mol/L ammonia-N"* L-asparagineapC 14.0 9.3 20.9 11.7 25.6 22.7 14.8 13.4 12.5

55.7 50.3 66.6 61.5 84.0 50.3 63.6 50.7 65.8

a Nonfasting blood sample. Single measurement. Average of two determinations.

aqueous NH4C1standards, are I 1 pmol/L. This represents a removal efficiency of 90% for the lowest plasma NH3-N level measured (sample no. 2).

ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983

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Table 11. Summary of Automated Plasma Asparagine Recovery Experiments amt of L-asparagine, mmol added" found 2.50 x 5.00 x 1.00 x 5.00 x 1.50 x

10-5 10-5 10-4 10-4 10-3

x x x x 1.86 x

2.30 4.76 9.72 5.07

8min

1

' O r II

% recovery

10-4

92.0 95.2 97.2 101.4

10-3

104.0

10-5 10-5 10-5

av

98.0

CALIB.

6

I

* 4.8

Standard aqueous aidditions into 2-mL aliquots of mmol of Lpooled plasma containiing 1 . 0 8 X asparagine. Average of three determinations. a

Figure 3 shows a tracing of a typical recording obtained from the L-asparagine assay of the same nine plasma samples. Values obtained from thie NH3-N and L-asparagine assays for these nine fresh plasma !samplesare summarized in Table 1. The values listed in Table I fall well within the expected normal range for fresh bl.ood plasma samples (NH3-N, 10-80 pmol/L (13);L-asparagine, 26-86 pmol/L, (12)). The normal endogenous NH3-N levlels shown in Table I would have introduced errors ranging between +18 and +45% in the I,asparagine assay of these samples if they were not removed prior to the enzymatic ;step. The specificity of the L-asparaginase enzyme used was tested. The only possible interferent reported for Lasparaginase from E. coli is the amino acid, L-glutamine (213. Aqueous standards of r,-glutamine and L-asparagine in the range between and mol/L were introduced into the system and assayed. The only L-glutamine standard that produced a response was mol/L (AI3 =: 13 mV; equivalent to approximately 2 x lo4 mol/L asparagine). Normal levels of glutamine in blood pla,sma are reported to range between 420 and 760 pmol/L (12). Due to the high specificity of the asparaginase for asparagine, glutamine at normal levels in plasma should not significantly interfere with the accuracy of the method. Indeed, for the normal range of glutamine expected, positive errors of only +1.5 to 3.0% would be observed for asparagine assays on samples containing the lowest normal levels of asparagine (Le., 26 pmol/L). Abnormally high levels of glutamine in th.e sample would present a more sig.nificant source of error. The accuracy of the imethod for handling blood plasma samples was further evaluated by performing analytical recovery studies on pooled plasma samples spiked with known amounts of L-asparagine. Table I1 summarizes the results of that study. It is evident from the data shown that the method has excellent recovery characteristics in the concentration range of interest. Finally, an attempt to evaluate the efficiency of the predialysis unit in removing NH3-N ranging between normal and abnormal levels in blood serum samples was undertaken. Figure 4 shows the results of this study. On the basis of the peak height values recorded with and without the use of the predialysis unit, it can be !seenthat the efficiency of the on-line predialysis unit increases as the NH3-N background in the sample increases. This is perhaps due to the variation in the diffusion rate of NH3 gas through the gas-permeable walls of the Teflon coil. Rates of gas transfer vary with concentration under controlled conditiolns; therefore, as the NH3-N concentration increases in the sample, so does its diffusion rate, resulting in more efficient NH3-N removal from the sample. At low NH3-N concentrations, diffusion is slower. The decreasing diffusion rates as the NH3-N concentration decreases in the sample and the relatively short residence time (nonequilibrium process) in the dialysis unit can account for the

i 1000

B

Flgure 4. Tracing of strip-chart recording illustrating the efficiencyof the on-line predilyzer unit in removing normal to abnormally high levels of NH,-N from spiked serum samples: (A) without dialysis unit; (8) with dialysis unit. Cslibrants are NH,CI standards in pmol/L concentrations. The base line potential for both A & B is the same.

almost pseudo-steady-state residual peak heights obtained. In practice, abnormally high background levels of NH3-N (up to approximately 550 pmol/L) can be effectively removed with this system and, therefore, offer no significant interference in the measurement of L-asparagine in its normal physiological range. For example, in the case of an extremely elevated ammonia-N sample, Le., sample no. 7 in Figure 4A (550 pmol/L ammonia-N), an error of only +11% would be found for the L-asparagine value if the sample contained the average normal level of asparagine. Fortunately for practical purposes, such elevated ammonia-N values are rare occurrences. In summary, we have reported here the development, evaluation, and application of an automated enzymatic assay system which utilizes an on-line gas dialyzer and potentiometric ammonia detection. The main advantage of the system is that the interference from variable background NH3-N levels present in physiological fluids is eliminated with the incorporation of the on-line predialysis unit. The tedious and time-consuming sample pretreatment methods currently used are not needed making this system attractive for routine clinical analysis. The system exhibits excellent detection limits, high precision, and good accuracy. While applied here only for the determination of asparagine in blood plasma samples, this system can be easily modified to assay other important amino acids and metabolites for which there exists selective deaminating enzymes and cells. Registry No. Ammonia, 7664-41-7; L-asparagine, 70-47-3; L-asparaginase, 9015-68-3.

LITERATURE CITED (1) Fung, K. W.;Kuan, S. S.;Sung, H. Y.: Guilbault, G. G. Anal. Chem. 1979. 5 1 . 2319-2324. (2) Guiibauit, 'G. G.; Chen, S. P.; Kuan, S. S. Anal. Lett. 1980, 13, 1607-1824. (3) Rechnitz, G. A.; Arnold, M. A.; Meyerhoff, M. E. Nature (London) 1979, 278,446-447. (4) Arnold, M. A.; Rechnitz, G. A. Anal. Chim. Acta 1980, 173, 351-354. (5) Kovach, P. M.; Meyerhoff, M. E. Anal. Chem. 1982, 5 4 , 217-220. (6) Kuriyama, S.;Rechnitz, G. A. Anal. Chim. Acta 1981, 131, 91-96. (7) DiPaolantonio, C. L.; Arnold, M. A.; Rechnitz, G. A. Anal. Chim. Acta 1981, 128, 121-127. (8) Hikuma, M.; Obana, H.; Yasuda, T.; Karulu, I.; Suzuki, S.Anal. Chim. Acta 1980. 116. 61-67. (9) Lienado, R: A.; Rechnltz, G. A. Anal. Chem. 1974, 4 6 , 1109-1112. (10) Mascini, M.; Rechnitz, G. A. Anal. Chim. Acta 1980, 116, 169-173. (11) Mascini, M.; Palleschi, G. Anal. Chlm. Acta 1982, 736, 69-76. (12) Ibbot, F. A. In "Ciinical Chemistry, Principles and Techniques", 2nd ed.; Henry, R. J., Cannon, D. C., Winkieman, J. W., Eds.; Harper and Row: Haggerstown, MD, 1974; Chapter 18.

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(13) Routh, J. I. I n “Fundamentals of Clinical Chemistry”; Tletz, N. W., Ed.; W. B. Saunders, Philadelphia, PA, 1976; Chapter 16. (14) Perry, T. L., Hansen, S. Clln. Chlm. Acta 1969, 25, 53-58. (15) Matthews, D. M.; Muir, G. G.; Baron, D. M. J . Clln. Pathol. 1964, 17, 150- 153. (16) Goodwin, J. F. Clln. Chem. (Wlnston-Salem, N . C . ) 1966, 14, 1080- 1090. (17) Constantsas, N. S.: Danelatau-Athanassladon, C. c//n, chlm. Acta 1964, 9 , 1-12. (18) Masclnl, M.; Gullbault, G. G. Anal. Chem. 1977, 49, 795-798. (19) Fratlcelii, Y. M.; Meyerhoff, M. E. Anal. Chem. 1961, 53, 992-997. (20) Meyerhoff, M. E.; Robbins, R. H. Anal. Chem. 1980, 52, 2383-2387.

(21) ”Biochemica Information 11”; Boehringer: Mannheim, 1975; pp 34-35. (22) Slgma Chemlcal Co. Catalogue, 1982, p 138.

RECEIVED for review August 26, 1982. Accepted November 12,1982. Acknowledgment is made to the National Institutes Health (Grant No- l-RO1-GM 28882-01) for support of this research.

Of

Membrane Electrode for the Determination of Actinyl(V1) Cations Peggy A. Bertrand, Gregory R. Choppin,” and Lln Feng Rao Department of Chemlstv, Florida State University, Tallahassee, Florida 32306

Jean-Claude G. Bunzll Institut de Chimie Minerale et Analytique, Universite de Lausanne, 1 105 Lgusanne, Switzerland

A coated wlre speclflc electrode has been developed for actlnyl(V1) cations. The electrode responds In Nernstlan fashlon to actlnyl(V1) concentratlons of 105-10-2 M between pH 2 and 5. The Interference by M(I), M(II), M(III), and MO,’ catlons is small even when [M(III)]:[AnO;+] 10. However, Th( I V) does Interfere. The electrode can be used NpO;’, and In complexatlon and redox studles of UO;,’ Pu02,+ wlthln Its useful range of pH and An02’’ concentration.

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Electrode membranes sensitive to UOZz+have been made by incorporation of uranyl compounds into organic matrices (1, 2). Recently Senkyr e t al. (3) reported U(V1)-sensitive ”neutral-carrier”/PVC/chloronaphthalenemembranes which are not constructed with macroscopic quantities of uranyl. Such membranes function by coordination of the neutralcarrier ligand t o the metal ion in solution. Because the membranes do not require the inclusion of significant amounts of metal ion, it seemed promising t o study their use with the more radioactive actinides such as plutonium. Electrodes for use with radioactive species should have certain features to make them practical; Le., they should be small and, if possible, should not incorporate significant amounts of the species. Both these constraints are met by electrodes constructed by using the coated-wire technique described by Freiser ( 4 , 5 ) . This paper reports on the development of a coated-wire electrode (CWE) by using the membrane system of Senkyr et al. and describes the response of these electrodes to actinide cations in oxidation states 111-VI. The voltage response of an electrode t o an ion in which i t is sensitive can be described by the Nernst equation a t constant ionic strength

E , = m log c

+ Eo

where E, is the voltage measured a t concentration c and Eo is the voltage for unit concentration. The slope m should have a value of 2.303RT/nF or 59.2 and 29.6 mvldecade change

in cation concentration for cation charges, n, of +1and +2, respectively. An experimental slope which agrees with the theoretical slope will be referred to in this paper as a “Nernstian” slope while a slope larger than the Nernstian value will be referred to as “over-Nerstian”. EXPERIMENTAL S E C T I O N Metal Solutions. Metal stock solutions were prepared by dissolving the nitrates or oxides in HCl or HC104. Working solutions were made by dilution of the stocks with appropriate amounts of aqueous NaCl or acetate buffer t o the desired metal concentration and 0.1 M ionic strength. The pH was adjusted with reagent grade NaOH and HC1 with a Beckman Model 1019 research pH meter equipped with a Corning combination pH electrode. The 2a7Npand 242Puoxides were obtained from Oak Ridge National Laboratory. Membrane Coating Solution. N,N’-Diheptyl-N,N’,6,6tetramethyl-4,8-dioxaundecanediamide(DTDD) was synthesized at the Institut de Chimie Minerale et Analytique and used without further purification. Poly(viny1 chloride) (PVC) from Aldrich was dissolved in tetrahydrofuran (THF) to prepare a stock solution (0.05 g/mL). The membrane coating solution was prepared by dissolving 0.026-0.039 g of the ligand in 6.0 mL of PVC/THF stock plus 0.46 mL of 1-chloronaphthalene. The 1-chloronaphthalene was added as a plasticizer and was necessary to produce a liquid membrane. The resulting viscous, cloudy solution separated into a clear yellow supernatant and a small amount of white gelatinous precipitate upon standing. Construction of Electrodes. One end of a coaxial cable (e.g., Belden RG-58/U) was stripped of 5 cm of its outer insulation and wire screening. At the end of the 5 cm, inner insulation was removed to expose 2 cm of bare copper wire. This was sanded until the end was flat and the surface no longer shiny. It was cleaned with soap and rinsed with distilled water and acetone. The membrane was applied by dipping the exposed end of the wire into the membrane coating solution to a depth of about 1.5 cm, taking care to avoid disturbing the gelatinous precipitate. This coated the wire with an organic film which was allowed to dry approximately 1 min prior to repeating the dipping procedure. In this manner, three to four membrane coats were applied to the wire. Following the final application of membrane/coating solution, the wire was air-dried 45-60 min and wrapped in parafilm so that only the small organic “bead”near the tip of the wire was exposed.

0003-2700/83/0355-0364$01.50/0 Q 1983 American Chemical Society