2586
Anal. Chem. 1984, 56,2586-2590
Homogeneous Potentiometric Enzyme Immunoassay for Human Immunoglobulin G Tekum Fonong and G . A. Rechnitz* Department of Chemistry, University of Delaware, Newark, Delaware 19716
Human IgG Is determined In a homogeneous Immunoassay method whlch utlllzes potentiometrlc CO, gasaenslng membrane electrodes. The method Is based on the inhlbltlon, by IgG, of CO, productlon from P-ketoadiplc acld catalyzed by chloroperoxldase (CI-POD) enzyme conjugated to IgG antibody. Under appropriate experlmental condltlons, the Inhlbitbn of the 1:l enzyme-antlbody conjugate Is shown lo be a function of IgG levels In the pg mL-' range with good selectlvtty. Separatlon of bound from unbound conjugate or the use of a second antibody are not required In thls method.
The impressive sensitivity of enzyme immunoassay (EIA) and the attractive selectivity of ion and gas-sensing potentiometric membrane electrodes (I) suggest that a combination of the two should offer a powerful tool for clinical or biochemical measurements (2) with particular advantages of simplicity if the C 0 2 or NH3 gas-sensing membrane electrodes could be employed. Homogeneous EIAs employing electrochemical techniques have been reported (3). Work reported to date on enzyme immunoassay with potentiometric membrane electrodes ( 4 , 5 )has been of the heterogeneous type, e.g., requiring a separation step or the use of a second antibody. The separation is necessary because the enzyme is active in the bound and unbound state. We now report a homogeneous potentiometric EIA for h u m m immunoglobulin G (IgG) based on enzymatic rate kinetics in which the separation of free and bound enzyme label is not necessary. The enzyme used in this study is chloroperoxidase (C1-POD). This enzyme catalyzes the bromination of P-ketoadipic acid in the presence of Hz02 and NaBr. Gaseous carbon dioxide is a product of the reaction, and it is detected by a COz gas-sensing electrode. Cl-POD is a glycoprotein (MW 42000) with a carbohydrate content of approximately 25-30%. The carbohydrate portion is not required for enzymatic activity. The enzyme can thus be covalently linked to other proteins (e.g., an IgG antibody) after mild oxidation of the carbohydrate portion. The major product of conjugation of C1-POD with human IgG antibody is a 1:l enzyme-antibody (E-Ab) conjugate which retains both the enzymatic activity and the immunochemical activity. The pH optimum of the enzymatic activity of the conjugate is shifted toward a higher and broader range than observed with the enzyme alone and suitable for immunoassay reactions. The conjugate loses the enzymatic activity on combining with the antigen IgG and the phenomenon can be used as the basis of a homogeneous immunoassay for the estimation of IgG. Figure 1 illustrates the overall reaction scheme used in this study. I t shows the C1-POD-catalyzed conversion of P-ketoadipic acid to products including the C 0 2 gas monitored potentiometrically with a pCOz membrane electrode a t p H 6.00. The E-Ab conjugate used for the study was the 1:l C1-POD-anti-(human IgG) IgG conjugate prepared by the method of Nakane and Kawaoi (6).
-
0003-2700/84/0356-2586$01.50/0
According to the scheme of Figure 1,the maximum rate of C02 liberation will be obtained when the reaction is initiated by addition of E-Ab conjugate to the substrate. If the E-Ab conjugate is first incubated with antigen (IgG) before reaction, the observed rate of C 0 2 liberation will be decreased @I&), the decrease in activity depending on the concentration of IgG incubated with the E-Ab conjugate. This effect serves as the basis for IgG antigen determination. It will be shown that the potentiometric enzyme immunoassay scheme of Figure 1 can be optimized to give analytical results for IgG determinations with good sensitivity and selectivity. The E-Ab conjugate is stable for a t least 1 month when stored a t 4 OC. Since the IgG is determined via direct inhibition of conjugate activity, no separation step or second antibody is necessary and the system meets the requirements of a potentiometric homogeneous immunoassay. The sensitivity of the method (in the pg mL-l range) is more that adequate for normal clinical levels of human IgG, so that very small samples can be used in practice.
EXPERIMENTAL SECTION Instrumentation. Incubation of enzyme-antibody conjugates with human IgG was carried out in 3-mL vials while enzymatic reactions were carried out in a 10-mL jacketed glass cell thermostated at 30 & 0.1 "C with a Haake Model FM constant-temperature circulator. Potentiometric measurements were made by using a Corning Model 12 Research pH/mV meter in conjunction with a Heath Schlumberger Model SR-255Bstrip chart recorder operated at 0.2 in./min and a range of 100 mV. Carbon dioxide liberated from the enzymatic reactions was monitored with an Orion Model 95-02 carbon dioxide membrane electrode. Reagents. Deionized water was used throughout in the preparation of solutions. Enzymatic reactions involving the native enzyme were carried out in 0.10 M glycine-H2S04,pH 3.00, buffer. Enzymatic reactions involving the enzyme-antibody conjugate were carried out in 0.10 M, pH 6.00, phosphate buffer. 0-Ketoadipic acid (catalog no. K-0500), 0-amylase from sweet potato (catalog no. A-8781),goat immunoglobulin G (catalog no. I-5256), chloroperoxidase (EC 1.11.1.10),lot no. 22F-02174, from the mold caldariomyces fumago (catalog no. C-0887),catalase (catalog no. C-lo), control serum and diluent (Type 1-A product no. C-7262), and Sephadex G-200 (particle size 40-120 pm) were purchased from Sigma Chemical Co., St. Louis, MO. The rabbit anti-(human IgG) (H & L chain) (lot. no E005, code no. 65-155, Control R 027) was purchased from Miles-Yeda Ltd., Israel, and human IgG (H & L chain) (lot no. 44, code no. 64-145, Control R 115) was purchased from Miles Laboratories, Elkhart, IN. Phosphate buffered saline (PBS),pH 7.2, and other reagents were prepared from reagent grade chemicals. A standard solution of 25 units mL-l chloroperoxidase was prepared in 0.10 M sodium phosphate solution, pH approximately 4.0, and this solution was stored at 4 "C until needed for experiments. The average enzyme shelf life was at least 1 month when stored at 4 "C. Reconstituted solutions of human IgG and anti-(human IgG) IgG were prepared in saline and distilled water, respectively. These solutions were stored at 4 and -20 "C, respectively. A stock solution of P-ketoadipic acid (0.125 M) was prepared in pH 6.00 phosphate buffer. Residual carbon dioxide can be removed from this solution by gently heating the solution at 40-60 "C and degassing; but stirring the solution vigorously for 10 min at room temperature was adequate for our purposes. 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 2587
p-ketosdlplc
+
H202 +
acid
+
Br-
Product8
+
IFb;l
H+
RI g 0 < R rn a X
Figure 1. Schematic representation of the (EC 1.1 1.1.10) chioro-
peroxidase-catalyzed liberation of carbon dioxide from @-ketoadiplcacid and the basis of the enzyme immunoassay. 100
100
80.
300 400 mU/mL
500
600
Figure 3. Calibration curve of chloroperoxidase activities as a function of initial rates, using 12.5 mM @-ketoadiplcacid at pH 3.00 and 30 O C .
A
.-
200
60, -10
CI
V
4
40. -30
\
;
20
-
" L
-50.
.-e
1
2.00
3.00
4.00
5.00
6.00
PH
Flgure 2. pH profile of chloroperoxidase-anti-(human IgG) IgG conjugate compared with the pH profile of the unconjugated chloroperoxidase enzyme at 30 O C .
Solutions of enzyme-antibody conjugate were stored at 4 "C until needed for experiments. Procedures. Determination of Rates of Halogenation of &Ketoadipic Acid. To the thermostated cell a t 30.0 f 0.1 "C was added 200 pL (0.220 M) of Hz02,100 ILL(6.25 X low2M) of 8-ketoadipic acid, 100 ILL(0.10 M)of NaF, and 1.40 mL of pH 3.00 glycine-HzS04buffer. The solution was stirred at a uniform rate until the pC0, electrode inserted in it reached a steady potential (-10 min). The reaction was then initiated by addition of 200 pL of chloroperoxidase enzyme (25 units mL-*), so that the total reaction volume was 2.00 mL. The initial rate of COz liberation was measured following established procedures (7). The procedure was repeated for NaC1, NaBr, and NaI. Determination of the pH Profile of Chloroperoxidase. Buffers in the pH range 1.50-4.00 were prepared from glycineH2S04mixtures, pH 5.00 from glycine-NaOH, and pH 6.00-7.00 from phosphate salts. To the thermostated cell at 30.0 0.1 OC was added 100 pL (0.10 M) of NaBr, 100 pL (0.220 M) of HzOz, 100 pL (6.25 X M) of 8-ketoadipic acid, and 1.45 mL of pH buffer. The reaction was initiated by addition of 250 pL (25 units mL-') of chloroperoxidase so that the total reaction volume was 2.00 mL. The initial rates of CO, liberation were recorded at each pH in mV/min. The results are shown in Figure 2. Preparation of the Chloroperoxidase Activity Calibration Curve. To the thermostated cell at 30.0 f 0.1 OC was added 100 ILL(0.280 M) of NaBr, 100 NL(0.220 M)of H202,and 200 ILL(0.125 M) of P-ketoadipic acid. A calculated volume of a pH 3.00 glycine-HzS04 buffer was then added to the mixture. When an aliquot of stock chloroperoxidase (1250 munits/mL) was added last, the final solution volume was 2.00 mL. The procedure was repeated for varying aliquots of the stock enzyme solution, the rate of reaction being measured each time in mV/min vs. the enzyme activity in munits/mL. The results are shown in Figure
*
3.
Preparation of the Calibration Curve for ,%Ketoadipic Acid. The stock solution of P-ketoadipic acid (0.105 M) was made
C
-70
* 0 n
/
-901
-130 10-3
10-2 @-ketoadipic
10'1 acld]
(M)
Figure 4. calibration curve for P-ketoadipic acid at pH 3.00 and 30 OC ushg 625 munlts of chloroperoxldase.
in a pH 3.00 glycine-HzS04buffer and stirred vigorously for about 10 min to expel any residual carbon dioxide gas. To the reaction cell at 30.0 0.1 OC was added 100 pL (0.10 M) of NaBr, 100 p L (0.220 M) of H202,500 pL (1250 munits/mL) of chloroperoxidase, and 2.00 mL of pH 3.00 buffer. Aliquots of stock Pketoadipic acid solution were added and the final steady-state potentials recorded as a function of concentration of P-ketoadipicacid. The results are shown in Figure 4. Preparation of the Enzyme-Antibody Conjugate. The method of Nakane and Kawaoi was used in coupling chloroperoxidase with antibodies to human IgG (6). The carbohydrate portion of C1-POD was used to form aldehyde groups by oxidation with sodium m-periodate (NaI04). Complete blocking of the aand t-amino groups of C1-POD with fluorodinitrobenzene (FDNB) prior to oxidation prevented self-coupling and allowed the C1POD-aldehyde to form Schiff bases with anti-(human IgG) IgG which has CY- and e-amino groups. NaBH4 was then added to stabilize the Schiff bases formed. The resulting conjugates are shown in the chromatogram of Figure 5. A slight modification involved oxidation of the enzyme by periodate for 15 min instead of 30 min. Chromatography of the E-Ab Conjugate on Sephadez G-200. After equilibration of a 60 X 1.5 cm gel-filtration column with PBS, a 3-mL solution containing E-Ab conjugates was placed at the top of the column, eluted with PBS at 12 mL/h, and 3-mL fractions were collected in 10-mL vials. The absorbance at 280
*
2588
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 100
Goat IgG
il
0.22.
0.18.
\
E 0
:0.14.
t
a
I
c. (D
n
a
0.10 1
0.5
0.021
2.0
2.5
Flgure 6. Callbration curve for human IgG using 300 munits of chloroperoxidase-anti-(human IgG) IgG at pH 6.00, 30 O C , and 12.5 mM
2;1
5
1.0 1.5 v g / m L 1gG
0-ketoadlpic acld. 10
15 20 Tube Number
25
30
Flgure 5. Elution profile of chloroperoxidase-anti-(human IgG) IgG conjugate compared with the elution profile of molecular weight markers (bars). Eluant was PBS (pH 7.2).
nm was recorded for each fraction on a Hitachi 100-80 spectrophotometer. One-milliliter aliquots of the molecular weight markers pamylase (15 mg/mL) and catalase (15 mg/mL) were prepared in PBS and chromatographed on the Sephadex G-200 column under identical experimental conditions as the E-Ab conjugates. The absorbance at 280 nm of the 3-mL fractions collected was also recorded. A plot of the absorbance vs. fraction number is shown in Figure 5. Determination of IgG. Several 1OO-bL aliquots of Cl-PODanti-(human IgG) IgG (-300 munits/mL C1-POD activity) were incubated with 1-20-bL aliquots of human IgG (1 mg/mL) in 3-mL vials at room temperature for 30 min. To the thermostated cell was added 200 fiL (0.125M) of P-ketoadipicacid, 100 pL (0.220 M) of H202,100 ML(0.280 M)of NaBr, and a calculated volume of pH 6.00 phosphate buffer so that when the enzyme-antibody/antigen complex was added last to the cell the final volume was 2.00 mL. Once a steady base-line potential was obtained, the reaction was initiated and the rate of C02liberation measured and recorded. The procedure was repeated for all the enzymeantibody conjugates incubated with antigen. The rates were converted to percent activity of enzyme in the E-Ab conjugate, assuming 100% activity when no IgG antigen was incubated with E-Ab conjugate. The percent activity of enzyme in the E-Ab conjugate was plotted as a function of the concentration of human IgG incubated with the E-Ab conjugate as shown in Figure 6.
RESULTS AND DISCUSSION Several different types of EIA have been used to quantify human IgG. They include competitive, immuno-enzymometric, sandwich (a), and immuno-precipitin (4)techniques. All of these assays fall in the category termed heterogeneous EIA. The focus of this study was to develop a more convenient homogeneous EIA technique based on potentiometric detection with the selective gas-sensing electrode for quantitation of human IgG. We have established that the C1-POD label on an IgG antibody can be detected homogeneously with a gas-sensing probe, the pCOz electrode. Antibodies to human IgG were coupled to Cl-POD enzyme (EC 1.11.1.10). The resulting E-Ab conjugate with a stoichiometry of 1:l (E/Ab) retained a high level of both enzymatic and immunochemical activity. Compared with the very narrow pH profile of the
native Cl-POD enzyme (91, the pH profile of C1-POD-anti(human IgG) IgG is broad so that 60% relative activity of C1-POD is retained a t pH 6.00 as shown in Figure 2. This fortunate change in the pH profile upon conjugation of the enzyme with antibody to IgG greatly facilitates the development of a homogeneous immunoassay in this case. The kinetic parameters influencing the activity of unconjugated C1-POD and its conjugate with human IgG antibody were first optimized by studying the influence of reagent concentrations, enzyme activities, pH, and reaction intervals. After the optimum conditions were established, human IgG was determined through its inhibitory effect on C1-POD activity when the latter was conjugated to anti-(human IgG) IgG. The inhibitory effect observed when antigen binds to the enzyme-labeled antibody may involve either steric hindrance of substrate access to the active site of the enzyme or induced conformational changes of the enzyme. Determination of Cl-POD Activity. Chloroperoxidase can utilize fluoride, chloride, bromide, or iodide to catalyze the formation of a carbon-halogen bond in the presence of a suitable acceptor molecule like P-ketoadipic acid (IO). It was found in this study that at halide concentrations of 5 mM the relative rates of halogenation of P-ketoadipic acid catalyzed by C1-POD are in the ratio of approximately 1:9:37:16 for F-, C1-, Br-, and I-. The iodination reaction rate is slower than the chlorination reaction rate because C1-POD can catalyze the peroxidation of iodide to iodine even in the absence of an acceptor molecule (IO). Thus, the reaction chosen for this study is the C1-PODcatalyzed conversion of 0-ketoadipic acid to 6-bromolevulinic acid and gaseous carbon dioxide. The reaction written as proposed by Morris and Hager ( 2 1 ) is shown in eq 1. -OOCCH2CH2C(=O)CHzCOO-
+ Br- + Hz02+
chloroperoxidase
2H+
(EC 1.11.1.10)
-OOCCH2CH2C(=O)CH2Br
'
+ 2H20 + C02
(1)
Figure 3 shows the effect of Cl-POD activity on the initial rate of C 0 2 liberation a t pH 3.00 and 30 "C. In order to establish optimum substrate, halide and H202concentrations, enzyme activity levels of 500 munits mL-l or higher were used since such activity levels are needed for maximum rates of COPliberation. It was necessary, however, to use an enzyme activity level in the linear region of the curve of Figure 3 for the immunochemical reaction, since measurements of human
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
IgG were based on the indirect measurement of C1-POD activity. Therefore, aliquots of a stock solution of C1-PODanti-(human IgG) IgG were taken in which the enzyme activity level was 300 munits mL-l, this being the upper limit of the linear region of the curve of Figure 3. The reagent concentrations were similarly optimized by varying one parameter while keeping other experimental conditions constant. The calibration curve for @-ketoadipicacid is shown in Figure 4. This plot shows the linear region to be on the high concentration side for the substrate @-ketoadipicacid (concentration >5 x M) with a slope of 54 mV/decade. The concentrations of substrate taken for the immunoassay studies in this study were chosen from the linear range of the Calibration curve of Figure 4. Purification and Characterization of E-Ab Conjugates, To purify the conjugates resulting from the conjugation reaction, the reaction solution was chromatographed on a 60 x 1.5 cm Sephadex G-200 glass column, eluting the conjugates with PBS (pH 7.2). The absorbance of 3.0-mL fractions at 280 nm was recorded. Two elution peaks at 12 and 36 mL were obtained as shown in Figure 5. These results indicate that two E-Ab conjugates had resulted from the conjugation reaction, the conjugate of the first peak being a minor component. In order to identify the two E-Ab conjugates eluted from the gel-filtration column, molecular weight markers were chromatographed on the Sephadex column under identical experimental conditions. If a molecular weight of 42 000 is assumed for C1-POD (11) and 150000 for anti-(human IgG) IgG, the 1:l and 2:l @/Ab) enzyme-antibody conjugates would have molecular weights of 192000 and 234000, respectively. Suitable molecular weight markers (P-amylase, MW 200000, and catalase, MW 230000) were chosen for chromatography on the Sephadex G-200 column. The elution profile of the molecular weight markers is also shown (as bars) in Figure 5. The molecular weight marker @-amylaseis eluted from the column at 33 mL. Three peaks were observed in the elution profile of catalase at 15,39, and 84 mL. The first peak at 15 mL contains catalase as a whole, while the second and third peaks contain multiple subunits of catalase. A subunit of catalase has a molecular weight of about 50000. In comparing the elution peaks in the chromatogram of the E-Ab conjugates with the peaks (bars) of the molecular weight markers, one easily confirms the minor peak to contain the 2:l (E/Ab) enzyme-antibody conjugate and the major peak a t 36 mL to contain the 1:l (E/Ab) enzyme-antibody conjugate. These results are consistent with previously reported work (12). Enzyme activity was confirmed in aliquots of E-Ab conjugate taken from both peaks of Figure 5. However, the enzyme activity of the 1:lE-Ab conjugate was 5 times higher than that of the 2:l E-Ab conjugate. The sum total of enzyme activity from the two conjugates was approximately equal to 80% of the initial enzyme activity of C1-POD before conjugation to antibody. This indicates that a high level of enzyme activity was retained through the conjugation process (6). Immunochemical Tests of the E-Ab Conjugates. Two 100-pL aliquots of the 1:l and 2:l E-Ab conjugate were incubated for 30 min at room temperature with and without 0.2 mg of human IgG. For the aliquots without human IgG, enzyme activity was determined to be 5 times higher for the 1:l E-Ab conjugate than the 2:l E-Ab conjugate. This indicates that most of the enzyme was bound in the 1:l E-Ab conjugate. For the aliquots incubated with human IgG, enzyme activity was shown to decrease to a greater extent by antigen-binding in the case of the 1:l E-Ab conjugate. Immunochemical activity was therefore also higher in the 1:l E A b conjugate. This is reasonable because when two enzyme
2589
molecules are coupled to one antibody molecule (as in the 2:l E-Ab conjugate), the antibody is sterically hindered from reaction with added antigen. As a result, enzyme activity in this conjugate is less attenuated by the presence of antigen in the system. On the basis of these results, only the 1:lE-Ab conjugate was used for subsequent experiments. Cross-Reactivity Tests. In place of human IgG, goat IgG was incubated for 30 min with the 1:l E-Ab conjugate. When this solution was added to a system containing reactants as shown in eq 1,it was found that the rate of liberation of carbon dioxide catalyzed by the E-Ab a t pH 6.00 was unaffected by up to 400 pg of goat IgG in the system. These results indicate that immunochemical selectivity is preserved and that selective measurements of human IgG can be made. Blank Experiments, Experiments were carried out to ascertain that the rate of decrease of enzyme activity of Cl-POD was due solely to the immunochemical reaction between human IgG and the anti-(human IgG) IgG coupled to C1-POD. In these experiments four 100-pLaliquots of Cl-POD (3 units mL-') were incubated for 30 min at room temperature, two with 400 pg of human IgG and two without human IgG. After incubation it was shown (according to reaction 1)that at pH 3.00 and p H 6.00 the rate of C 0 2 liberation from @ketoadipic acid was not affected by the presence of human IgG in the system. Evidence of Operation of Homogeneous EIA. Three 100-pL aliquots of the 1:l E-Ab conjugate were incubated for 30 min at room temperature without human IgG (case l), with 5 pg of human IgG (case 2), and with 5 pg of human IgG + 2 pg of anti-(human IgG) IgG (case 3). After incubation each aliquot was added in turn to a solution system as in Figure 1,containing excess P-ketoadipic acid. The rate of C 0 2 liberation was measured in each case. The highest rate (3.5 mV/min) was observed in case 1 where no human IgG was present. The second highest rate (1.4 mV/min) was observed in case 3, and the lowest rate (0.9 mV/min) was observed in case 2. These results show that in the case where anti-(human IgG) IgG is present in solution, it competes with the anti(human IgG) IgG conjugated to C1-POD for the human IgG antigen in the system. This competition effectively minimizes the human IgG's attenuating effect on the enzyme and a higher rate of enzyme activity is obtained in case 3 in comparison to case 2; this pattern applies to a homogeneous system such as that presented in Figure 1. Analytical Determination of Human IgG. In order to obtain a quantitative measure of the material to be determined, IgG, the reaction scheme of Figure 1must be operated under kinetic conditions where the analyte is rate-limiting. This requires that the reactants of eq 1 be present in excess. Since human IgG exerts its influence by inhibiting the catalytic activity of the enzyme-antibody conjugate, it is convenient to plot the measured IgG concentrations vs. the percentage of conjugate activity with 100% corresponding to no inhibition. Such a calibration curve is given in Figure 6 where the selectivity for human over goat IgG is also shown. It should be noted that such a presentation corresponds to a first-order decay process, so that a replotting of the data on a logarithmic scale will give a straight-line calibration plot convenient for analytical use. The sensitivity of the method is well below the normal clinical range for human IgG (8-14 mg/mL). This means that very small samples could be used in practice or that samples could be heavily diluted to minimize matrix effects. Table I summarizes recovery and precision data for IgG determinations in the pg/mL range for ten replicate measurements. We found no differences in recovery or precision data when the rate measurements were carried out in pooled human control serum instead of aqueous buffers. Although actual
2590
Anal. Chem. 1984, 56, 2590-2592
Table I. Precision and Recovery Studies of Human IgG IgG added, pg mL-l
recovery
within-run precision, % RSD
0.4 0.6 1.0 1.5 2.0 2.5 3.0 3.5
98 98 97 96 94 97 97 96
k5.0 14.5 k3.6 k2.6 h2.0 k2.4 k1.5 k1.0
%
97% av
Chem. 1983, 55, 202R-214R. Wehmeyer, K. R.; Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Methods Enzymol. 1983, 9 2 , 432-437. Alexander, P. W.; Maltra, C. Anal. Chem. 1982, 5 4 , 68-71. Gebauer, C. R.; Rechnitz, G. A. Anal. Blochem. 1982, 124, 338-348. Nakane, P. K.; Kawaoi, A. J . Hlstochem. Cytochem. 1974, 22, 1084-109 1. Meyerhoff, M. E.; Rechnltz, 0. A. Anal. Blochem. 1979, 9 5 , 483-493. Wisdom, 0. G. Ciin. Chem. (Winston-Salem, N . C . ) 1976, 2 2 , 1243-1 255. Shew, P. D.;Hager, L. P. J . 8/01. Chem. 1961, 236, 1626-1630. Hager, L. P.; Morrls, D. R.; Brown, R. S.; Eberweln, H. J. 8lol. Chem. 1986, 241, 1769-1777. Morrls, D. R.; Hager, L. P. J . 8/01.Chem. 1966, 1763-1768. Boorsma, D.M.; Kalsbeek, G. L. J . Hlstochem. Cytochem. 1975, 2 3 , 200-206.
patient samples are not available to us, we expect that this method could be useful for clinical purposes. LITERATURE C I T E D (1) Arnold, M. A.; Meyerhoff, M. E. Anal. Chem. 1984, 56, 20R-48R. (2) Davis, J. E.; Solsky, R. L.; Glerlng, Linda; Malhotra, Saroj. Anal.
RECEIVED for review June 12,1984. Accepted August 3,1984. The support Of the National Institutes of Health (Grant GM-25308) is greatly appreciated.
CORRESPONDENCE Thermospray Liquid Chromatographic Interface for Magnetic Mass Spectrometers Sir: The thermospray technique (1)is becoming recognized as a practical approach for on-line LC-MS. Up to now thermospray interfaces have been used on quadrupole mass spectrometers in which the ion source is a t or near ground potential. Recently it has been demonstrated that thermospray provides a "soft" ionization technique capable of producing intact molecular ions from large, nonvolatile molecules. Exploitation of this technique for large molecules has been limited by the mass range and resolution available from the quadrupoles presently used. Although the mass range available from commercial quadrupole instruments has increased substantially in recent years, it appears that magnetic deflection instruments will continue to enjoy a distinct advantage over quadrupoles in high-mass and high-resolution capabilities. In the thermospray system described earlier (1-4), the vaporizer and its associated control system must reside a t essentially the same potential ass the ion source, which in typical instruments is several kilovolts off ground. Also, the pressure in the source pump-out line is typically a few torr, a pressure range in which gas discharges are easily initiated at potential differences of a kilovolt or less. While it is possible to float the entire LC-MS interface a t ion source potential, it is desirable for operator safety and convenience to keep the LC and the mechanical vacuum pump at ground. The system developed to provide a practical solution to the problems associated with operating the thermospray interface on a magnetic mass spectrometer is shown schematically in Figure 1. The major portion of the interface is identical with that described earlier (I), but the necessary measures have been taken to deal effectively with the problems introduced by operating the ion source at high voltage. The LC effluent is coupled to the thermospray vaporizer by using a length of 150-pm-i.d. fused silica capillary (SGE,Austin, TX). The source is pumped by a glass dry iceacetone cooled trap backed by a 300 L/min mechanical vacuum pump. The line from the
source to a trap is a 1.2-cm-i.d. stainless steel tube which passes through the source housing by means of an electrically isolated flange. The LC connection and heater and thermocouple connections pass through a similar electrically isolated flange on the opposite side of the source housing. A discharge suppressor is installed inside the rubber hose connecting the trap to the mechanical vacuum pump. This device is similar to one described earlier by Wojcik and Futrell5 for coupling a chemical ionization source at high voltage to a grounded inlet system. The present discharge suppressor consists of 11disks of perforated stainless steel which are separated and supported by 2.5 cm long ceramic standoffs. Eleven 10-MO 2-W resistors are connected in series to this stack of discs, and the whole assembly is installed inside the 4-cm-i.d. rubber hose connecting the trap to the mechanical vacuum pump. The upper end of the resistor chain is connected to the ion source potential and the lower end to the grounded vacuum pump. The temperature controller for the thermospray system is similar to that used previously for the quadrupole interfaces, but it is mounted on high voltage standoffs inside a grounded outer case. The necessary controls are brought out on insulated shafts through a clear plastic panel which allows the temperature controllers and readouts to be monitored while protecting the operator from contact with the controller operating a t ion source potential. Power is supplied through an isolation transformer with insulation rated for 4 kV. This system has been successfully operated with a variety of solvents including 0.1 M ammonium acetate at ion source voltages to 3.5 kV, the maximum available with the power supply available with the present instrument. In initial trials, total ion currents in excess of A into the magnetic analyzer were obtained by direct thermospray ionization using the above buffer. The mass spectra obtained were essentially identical with those obtained under comparable operating conditions by using the quadrupole. Results of experiments on the current flow through the
0003-2700/84/0356-25~0$0 I .50/0 0 lg84 American Chemlcal Society
