2332
Anal. Chem. 1980, 52, 2332-2336
be necessary to examine a sufficient segment of the mass spectrum of the suspect residue to rule out the presence of PCMBs (by the absence of fragment ion clusters resulting from losses of CH3, CHSCO, and CH3C0 + 2C1 from the M+ cluster) and other potential interferences not yet observed.
ACKNOWLEDGMENT The authors thank the following members of the Food and Drug Administration for their contributions to this report: Peter A. Dreifuss, Samuel W. Page, Albert M. Gardner, John W. Butler, James D. Link, William J. Trotter, and Lee J. Miller.
LITERATURE CITED (1) Shadoff, L. A.; Blaser, W. W.; Kocher, C. W.; Fravel, H. G. Anal. Cbem. 1978, 50, 1588-1588. (2) Crummett, W. B.; Stehl, R. H. EHP, Environ. Health Perspect. 1973, 5, 15-25. (3) Camoni, I.; DiMuccio, A.; Pontecorvo, D.; Vergori, L. J. Cbromatogr. 1978, 153,233-238. (4) diDomenico, A.; Merll. F.; Boniforti, L.; Camoni, I.; Di Muccio, A,; Taggi. F.; Vergori, L.; Colli, G.; Elli, G.; et al. Anal. Chem. 1979, 51,735-740. (5) O'Keefe, P. W.: Meselson, M. S.; Baughman, R. W. J. Assoc. Off. Anal. Cbem. 1978, 67, 621-626. (6) Shadoff, L. A.; Hummel, R. A. Bbmed. Mass Spectrom. 1978, 5, 7-13. (7) Harless, R. L.; Oswald, E. 0. 24th Annual Conference on Mass Spectrometry and Allied Toplcs, San Dlego, CA, May 9-13, 1976; pp 26-28.
(8) Harless, R. L.; Oswald, E. 0. 25th Annual Conference on Mass Spectromeby and Allied Topics, Washington, DC, May 29-June 3, 1977; pp 592-594. (9) Firestone, D. J. Agric. Food Cbem. 1977, 25, 1274-1280. (10) Firestone, D.; Clower, M.. Jr.; Borsetti, A. P.; Teske, R. H.; Long, P. E. J. Agric. Food Cbern. 1979, 27, 1171-1177. (1 1) Clark, F. R. S.; Norman, R. 0. C.; Thomas, C. B. J. Chem. Soc., Perkin Trans. 11975, 121-125. (12) Cadogan, J. I. G. J. Cbem. SOC. 1982, 4257-4258. (13) Baughman, R.; Meselson, M. EHP, Environ. Health Perspect. 1973, 5 , 27-35. (14) Bowes, G. W.; Mulvihill, M. J.; Simoneit, B. R.; Burlingame, A. L.; Risebrough, R. W. Nature (London) 1975, 256, 305-307. (15) Safe, S.; Hutzinger. 0. "MassSpectrometry of Pesticides and Pollutants"; CRC Press: Cleveland, OH, 1973; pp 72-73. (16) Jansson, B.; Sundstrom, G. Biomed. Mass. Spectrom. 1974, 1 , 389-392. (17) Baughman, R.; Meselson, M. I n "Chlorodioxins-Origin and Fate"; Blair, E. H., Ed.; American Chemical Society: Washlngton, OC, 1973; pp 92-104. (18) Herbst, E.; Scheunert, I.; Klein, W.; Korte, F. Chemosphere 1978, 7, 22 1-230. (19) Sundswom, G.; Hutzinger, 0.; Safe, S. Chemosphere1978, 5,267-298. (20) Maass, W. S. G.; Hutzinger, 0.; Safe, S. Arch. Environ. Contam. Toxicol. 1975, 3 , 470-478. (21) Lay, J. P.; Klein, W.; Korte, F. Chemosphere 1975, 4 , 161-168.
for review October l7, l979. Accepted September 2, 1980.
Continuous-Flow Analysis for Uric Acid in Biological Fluids, with Immobilized Uricase in a Closed-Loop System Asfaha Iob and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
Relatively Inexpensive and/or stable enzymes can be directly used in solution for repetitive determinations in closed-flow systems. Important ciinlcal determlnatlons (e.g., uric acld In bloiogical fluids), however, require enzymes that are not sufflclently stable and/or inexpensive to be used in solution for relatively long periods of tlme and at the high actlvlty levels needed for success In unsegmented closed-flow systems. Thls paper reports on the chemlcal Immobilization of uricase on controlledpore glass, certain characterlstics of the lmmobllized enzyme preparation, and application of it as packing In mixing-delay coils for the determination of uric acld In blologlcal fluids. Determinations can be peformed at a 100 samples/h rate with satlsfactory precision (24% RSD). The method was compared wlth a regularly used coiormetric procedure in the SMA 18/90 analyzer [correlation coefficient = 0.98 for 22 samples of human blood serum]. The lmmoblllzed enzyme preparatlon retains over 70 % of its initial activity afler repetltive use for more than 10 months.
The use of enzymes as analytical reagents has increased in recent years. As biological catalysts which work in complex living systems, enzymes offer two characteristics of paramount importance in analytical chemistry: (1) generally high selectivity (and occasional specificity); (2) capability of selfregeneration via the catalytic cycle. The first of these properties is widely recognized and used; the second usually restricts the use of enzymes to immobilized ones. Recently (I), however, we have shown that if the enzyme is relatively 0003-2700/80/0352-2332$0 1 .OO/O
inexpensive and relatively stable [so that it retains its activity toward the substrate of interest a t reasonable constant level at room temperature and with time] it can be directly used in solution for repetitive determinations in closed-flow systems; the closed loop affords enzyme recycling and reutilization. For enzymes relatively inexpensive and stable this approach has definite advantages over systems utilizing immobilized enzymes packed in chromatographic-type columns inserted a t a given point (before detection) in the flow train (21, namely, simpler and faster determinations. Unfortunately important clinical determinations employ enzymes that are not sufficiently stable and/or inexpensive to be used in solution for relatively long periods of time and at the high levels required for success in flow systems. For the development of determinations competitive in cost and number of determinations per hour in almost unattended continuous-flow analysis, enzymes such as uricase [used for specific uric acid determination in biological fluids] must be rendered stable and present in high local concentration. Immobilization, in these cases, offers a real advantage. This journal has devoted regular attention to the analytical applications of immobilized enzymes in the form of feature articles (3-5). There are four principal methods for enzyme immobilization: (1) containment by membrane; (2) entrapment; (3) adsorption; (4) covalent bonding. Immobilization by covalent bonding is best for continuous-flow analysis and either packed-bed reactors (6, 7) or open tubes with enzymes immobilized on the wall (7-9) can be used, In the case of packed beds, because of its mechanical stability, controlled-pore glass (and other types of porous glass beads) constitutes a convenient matrix for enzyme immobilization. This paper describes the immobilization of 0 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
2333
uricase (urate oxidase) by perhaps the most widely used approach to covalent immobilization (IO),involving the use of alkylamino glass and glutaraldehyde. T h e controlled-pore glass bearing immobilized enzyme was utilized to pack a delay-mixing coil which, with an instrument setup like one already described ( I ) , was used for the determination of uric acid in biological fluids [blood serum and urine]. EXPERIMENTAL SECTION Reagents and Solutions. All chemicals used through the work were of reagent grade. Uricase (E.C. 1.7.3.3) was obtained from Sigma Chemical Co. (St. Louis, MO) and was derived from hog liver (20 units/g of solid) or from Candida utilis (5.4 units/mg of protein). One unit of uricase activity will convert 1pmol of uric acid to allantoin/min at pH 8.5. Determination of the uricase units was accomplished following the supplier's directions ( I I ) . Deionized water was found satisfactory for solution preparation. Typical reservoir solutions consisted of 200-mL volumes of a borate-ammonium sulfate buffer [pH 9.5, 0.10 M in total ammonium and total borate] containing 15-20 mg of catalase (E.C. 1.11.1.6) (from bovine liver, lo5 units/mg; 1 unit = amount of catalase decomposing 1.0 pmol of Hz02/minat pH 7.0 and 25 "C while the HzOzconcentration falls from 10.3 to 9.2 pmol/mL of reaction mixture). Catalase was also supplied by Sigma. Apparatus. Determinations were carried out with an instrumental setup like one described earlier ( I ) . The coil was packed with immobilized enzyme on controlled-pore glass in place of the open one used for determinations with soluble enzymes. The peristaltic pump was positioned just before the point of sample introduction. Sample injection was performed with a rotatory valve constructed as previously described. (12). Typical glass coils f i e d with immobilized enzyme had an inside diameter of 1.5 mm and a length of 40 cm (12 turns, 4 mm 0.d.). The controlled-pore glass (CPG) was CPG-3000 from Electro-Neuclonics, Fairfield, NJ [80/100 mesh, 2869-A mean pore diameter, and 8.9 m2/g surface area] of the type used for glass permeation chromatography. Typically 0.7 g of the CPG-3000 with immobilized enzyme (about 7 U/g) was present in each coil; coils can be connected in tandem to increase coil length and extent of reaction if needed. Several trial-and-error loadings of the tubes have shown that best results are obtained when a straight tube with one end temporarily closed is loosely packed with the CPG and then coiled, one end being kept open. The temperature used for coiling did not affect the characteristics of the CPG chips. Immobilization is then accomplished by circulating the needed reagents in a closed loop with the help of a peristaltic pump. Immobilization of Uricase on CPG-3000.Covalent bonding of the enzyme to the inert matrix involved the preparation of alkylamino glass and reaction with glutaraldehyde. Although the chemistry of these reactions is not well understood, the individual steps include the reaction of CPG-3000 with 3-aminopropyltriethoxysilane (Aldrich Chemical Co., Inc., Milwaukee, WI), modification of the alkylamino glass by reaction with glutaraldehyde, and final incorporation of the enzyme. The actual procedure consisted of the following steps: (1)the glass coil packed with CPG-3000 was heated at 500 "C for 6 h; (2) after the coil was cooled to room temperature, 50 mL of 2% solution of 3aminopropyltriethoxysilane in acetone was continuously circulated through it for 10-20 min at a rate of 8 mL/min; (3) after silanization the coil was dried at 45 "C for about 48 h; (4) 100 mL of a 1% aqueous solution of glutaraldehyde was circulated through the dried aminoalkyl glass preparation for 30 min at a rate of 8 mL/min; (5) after treatment with glutaraldehyde the preparation was washed with deionized water; (6) 5-10 units of uricase in 10 mL of borate buffer of pH 9.4 was finally circulated through the coil at a rate of 2 mL/min for 24 h at room temperature; the excess enzyme solution was washed out with borate buffer followed by a 1.0 M NaCl solution in borate buffer and borate buffer again. This method of immobilization is an adaptation of the one originally reported by Robinson et al. (13). The units of uricase not immobilized were determined (11)in order to estimate the number of units immobilized. R E S U L T S AND DISCUSSION Clinical method development for uric acid determination
8.5
7.5
9.5
10.5 PH
Flgure 1. pH activity profile of uricase immobilized on C P G 3 0 0 0 .
is centered on enzymic conversion of uric acid to allantoin by action of uricase according to the following overall reaction: urate O2 H 2 0 allantoin H202+ C 0 2 (1) This reaction is specific and permits development of methods free from interferences that plague the nonenzymatic procedures: turbidity; reducing agents; many antibiotics. T h e preparation, purification, and properties of this cuproprotein were reported by Mahler et al. in 1955 (14). As discussed ( I ) , the presence of catalase yields the following overall reaction:
+
urate
+
-
+ 1/202uricase + catalase
+
allantoin
+ COz
(2)
which can be monitored by measuring amperometrically the decrease in oxygen level. Uricase has been successfully immobilized and used in some uric acid determinations. Nanjo and Guilbault, for instance, developed an enzyme electrode consisting of a platinum disk in contact with a layer of glutaraldehyde gel in which the uricase was immobilized (15). Hinsch e t al. coimmobilized aldehyde dehydrogenase and uricase on nylon tubes (16) and Murachi et al. reported the immobilization of uricase on alkylamine glass (17);in both of these reports the immobilized preparation was used for uric acid determination in segmented-continuous-flow analysis. Several functional characteristics of the immobilized uricase as described in the experimental part have been evaluated. First of all, two types of CPG glass chips were used. These were CPG-500 [mesh size 200-400; mean pore diameter 544 A; surface area: 57 m2/g] and CPG-3000 [mesh size 80-120; mean pore diameter 2870 A; surface area 9 m2/g]. Better flow characteristics were obtained with the CPG-3000 without a significant loss in sensitivity, and so the bulk of the work reported here was performed by using uricase immobilized on CPG-3000. pH Profile for Uricase Immobilized on Silanized Glass Chips. Experiments were conducted to establish the optimum pH, under flow conditions, for the determination of uricase. Figure 1 evaluates the p H variable in terms of peak height; return to base line was practically unaffected by pH changes. It can be concluded that maximum activity of the immobilized preparation occurs at a pH close to 9.5 and this p H has been used throughout the work reported here. Long-Term Stability of Uricase Immobilized on Silanized Glass Chips. Figure 2 shows a comparison of the stability of a soluble enzyme preparation and an immobilized uricase sample prepared from the same enzyme solution. The initial activity of the enzyme, in both solution and immobilized on controlled-pore glass, was taken as 100 arbitrary units.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
2334
‘““li‘s--___
0
h
IO+
’
@\ 2
4
6
0 0
,b
1‘1
14
Ib
/*
lo
.)+
DAYS
Figure 2. Operational stability CPG-3000.
(long term) of uricase immobilized on
Between assays both preparations were kept at 4 “C in sodium borate-ammonium sulfate buffer of pH 9.4. It can be seen that even after 10 months the immobilized enzyme retained more than 70% of its original activity while the enzyme in solution had lost more than 90% of its original activity in about 10 days. Even if it was stored in dried form, a t 4 “C, retention of activity by the immobilized preparation was found to be very satisfactory for analytical purposes. The same was observed with preparations subjected to solutions of different p H for different lengths of time; return to p H 9.5 restored optimum performance without loss in activity. A p p a r e n t Michaelis-Menten Constant. The Michaelis-Menten constant for soluble uricase [pH 8, 20 “C] is reM urate (18). By utilization of UV ported as 1.7 X monitoring to follow the uric acid conversion and conventional plots to estimate the Michaelis-Menten constant, K,, the soluble preparation in a buffer of pH 9.4 and 25 “C yielded values between 1.1and 1.5 X M for different batches of enzymes. The immobilized enzyme gave lower values for the apparent Michaelis-Menten constant, km(app); these were between 6.0 and 8.0 X lo4 M urate, depending on the preparation. It is known that immobilization may alter the Michaelis-Menten constant; the effect, however, generally results in an increase instead of the decrease that was observed here. Endo et al. (17) report values of 2.6 and 4.8 X lo4 M urate for km(app)and also observed a decrease in the MichaelisMenten constant upon immobilization of uricase on silanized glass chips. The lower value of this constant for immobilized preparations indicates that “saturation” [rate independent of urate concentration] occurs a t smaller concentrations of urate and thus limits the concentration range amenable to determination. On the other hand, rates (initial) are larger with smaller values for the constant, allowing one to obtain larger signals and detect them at earlier stages of the reaction. In any event, and as shown later in this paper, the decrease in k , in this case does not affect the performance of the flow system, and amperometric detection, enough to impair the determination of uric acid in biological fluids. T h e Determination of Uric Acid by Use of Immobilized Uricase o n CPG-3000 as P a c k i n g of Delay-Mixing Coils. Normal levels of uric acid in human beings are reported as 3.8-7.1 mg/100 mL and 2.6-5.6 mg/100 mL in serum of adult males and females, respectively (19). Normal values in children are between 2.0 and 5.5 mg/100 mL of serum (19). T h e normal range of uric acid in urine is 250-750 mg in 24 h (20). Low limits of detection are then not required for the determination in urine but are desirable for that in human blood serum in part owing to limitation in available sample size. A useful analytical procedure should provide linear calibration curves that extend below 2 mg of uric acid/100
1
min
Flgure 3. Typlcal transient signals obtained by injection of uric acid samples containing the concentratins indicated in the figure. Recorder span was 1 mV. Chart speed was 1 in./min.
mL of serum and beyond 8 mg/100 mL. Linear calibration curves with near zero intercept were obtained between 0 and 10 and 10 and 100 mg of urate/100 mL of solution. They were prepared from “Calibrate” serum calibration references (General Diagnostics,Division of Warner-Lambert Co., Morris Plains, NJ). Aqueous standard solutions gave slightly higher signals than the “Calibrate” reference samples. Typical signal profiles are shown in Figure 3. Linearity in the calibration plots extends into bulk concentrations larger than k,; this may be in part because the catalyzed reaction is very rapid compared to the rate of diffusion of substrate and in part because of the nature of the flow system-mixing coil combination. As the “plug” advances along the coil, substrate solutions of lower and lower concentrations are mixed with “fresh” enzyme. Compartmentalization of enzyme-substrate similar to that in a closed batch system is not realized here and when the “plug” reaches the detector a sort of integrated effect is realized instead of a measurement of the initial velocity utilized to extract the k , value. It is of interest to note that linear calibration at concentrations greater than k , have also been observed by Me11 and Maloy for an amperometric enzyme electrode of glucose oxidase immobilized in a polyacrylamide gel (21). The effect of flow rate on peak height and on the time for return to base line, th, is shown in Figure 4. A flow rate of 7 mL/min is recommended to realize good sensitivity and maximum affordable determination rate (about 100 determinations/h under these conditions). A smaller coil (three turns) was used in these studies without observing a significant decrease in signal height. The typical sample size used for
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
‘\
2335
10
46
e
9
e e
a
7
n
e
e
e 6
* e
Y
E 1
2
3 FLOW
4
5
6
7
v1 Y
1 1 1 1 . mr/min
Flgure 4. Effect of flow rate on signal peak height and time to return
v
to base line, fhs:
a
(0)fhs;
(0) peak height.
Table I. Effect of Injectea Sample Size o n Some Parameters of Analytical Relevancea sample size, W L 30 35 40 60 80 100
height, mm
return to base line, min
64.3 70.2 84.6 89.7 101.6 121.6
0.7 0.7 0.7 0.8 1.0 1.2
peak
e
5
6
Data are for the repetitive injection of a solution of uric acid, 10 mg/100 mL. Flow rate, 5 mL/min. determinations was 80 pL of a 10 mg/100 mL solution, since it provides reasonable sensitivity without much sacrifice in the time return to base line that dictates the determination rate. Table I shows data obtained with different sizes of loops inserted in the rotatory valve. The time for return to base line was observed to be independent of concentration. Reproducibility of the results was tested by repetitive injection of 80-pL samples of solutions containing 4 and 9 mg or uric acid in 100 mL of solution a t an injection rate of 110 samples/h. The relative standard deviation for sets of 30 injections (without rejecting any value) ranged from 2 to 4%. Interferences with the enzyme-catalyzed reaction are documented in the literature (22) and will not be discussed here. Interferences with the amperometric technique have also been discussed earlier (1, 23). In the repetitive determination of uric acid, allantoin [5-ureidohydantoin] accumulates as a product. No interference from allantoin (Aldrich Chemical Co.) was detected, however, even a t concentrations as high M, a level unlikely to be reached in practical as 1 X applications of the method. Uric acid levels in human blood serum were determined by the proposed method and with the commercial analyzer “SMA 18/90” [AutoAnalyzer 11, Technicon Instruments Corp., Tarrytown, NY] which utilizes the phosphotungstic acid method and is routinely used a t a local hospital. Figure 5 graphically illustrates the results for 22 samples. The Pearson correlation coefficient was found to be r = 0.98 with the SMA method yielding generally slightly higher results than the immobilized enzyme, unsegmented, continuous-flow determinations in closed-loop configuration. The phosphotungstic acid method is a nonspecific one (24) and has been reported to lack constant proportionality between the color yielded and the amount of uric acid present (25). The main limitation in the setup presented here is that high flow rates or gravitational flow [as used in the work of ref 13 are not possible because of the packing. Up to now, the same enzyme preparation has been used for more than 10 months to analyze serum samples without detection of deterioration
4
e
n
--
e e
.
.e e
Y
H
3
d
m
0
E
I
e
2
1
1
2
3
4
5
6
7
8
9
IO
SMA 1 8 / 9 0 METHOD
Flgure 5. Comparison of results obtained by sample injection in closed flow-through system and immobilized uricase and by the “SMA 18/90 Method”. Values in xand yaxis are in mg of urate/100 mL solution. Slope obtained by linear regression is 0.90, intercept is -0.11 mg/100 mL, r = 0.98.
of its performance. As many as 300 samples, of 80 KL of serum each, have been injected in a reservoir solution of 200 mL without producing interferences. In this case saving of the reservoir solution is not particularly important since the expensive reagent (the enzyme) is not present. A few determinations of uric acid in samples of human urine indicate that such an analysis can be performed even without dilution. The selection of shorter mixing-delay coils and the fact that the linearity of the calibration curve extends within the concentration range present in human urine allow adaptation of the procedure for the repetitive determination of uric acid in this biological fluid.
ACKNOWLEDGMENT The authors are indebted to Elaine Mills and the Stillwater Municipal Hospital for samples of human blood serum and for the results with the SMA 18/90.
LITERATURE CITED (1) Wolff, Ch-M.; Mottola, H. A. Anal. Chem. 1978, 50,94-98. (2) Bergmeyer, H. U.;Hagen. A. Fresenius’ Z . Anal. Chem. 1972, 26, 333-336. (3) Weetali, H. H. Anal. Chem. 1974, 46, 602A-815A. (4) Bowers, L. D.; Carr, P. W. Anal. Chem. 1976, 46, 544A-559A. (5) Gray, D. N.; Keyes, M. H. Anal. Chem. 1977, 49, 1067A-1078A. ( 6 ) Schifreen, R. S.; Hanna, D. A.; Bowers, L. D.; Carr, P. W. Anal. Chem. 1977, 49, 1929-1939. (7) Attlyat, A. S.: Christian, 0. D. Ana&st(London) 1080, 705,154-160. (8) Leon, L. P.;Sansur, M.; Snyder, L. R.; Horvath. C. Clln. Chem. (Wlnston-Salem, N.C.) 1977, 23 1556-1562. (9) Leon, L. P.; Chu, D.K.; Snyder, L. R.; Horvath, C. Clln. Chern. (Wlnston-Salem, N . C . ) 1980, 26. 123-129. (10) Chlbata, I. “Immobllized Enzymes”; Halsted Press (Printed by Kodansha Limited, Tokyo, Japan): New York, 1978. (1 1) SIGNA Technical Bulletin No. 292JJV, reissue; Sigma Chemlcal Co.: St. Louis, MO, June 1977. (12) Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56, 877-880. (13) . . Robinson, P. J.: Dunnill. P.: Liilv. M. D. Blochlm. Bloohvs. . . Acta 1971. 242, 659-661. (14) Mahler, H. R.; HUbscher, G.; Baum, H. J . Blol. Chem. 1955, 276, 625-641.
2336
Anal. Chem. 1980, 52, 2336-2338
(15) Nanjo, M.; Guiibauit, G. G. Anal. Chem. 1974, 4 6 , 1769-1772. (16) Hinsch, W.; Antonijevic, A.; Sundaram, P. V. Fresenius' 2.Anal. Chem. 1980, 307, 161. (17) Endo, J.; Tabata, M.; Okada, S.; Murachi, T., Clln. Chim. Acta 1979, 95,
(23) Woiff, Ch-M.; Mottola. H. A. Anal. Chem. 1977, 49, 2118-2121. (24) Musser, A. W.; Ortigoza, C. Am. J . Clln. Pathol. 1966, 45. 339-343. (25) Domagk, 0.F.; Schlicke Anal. Blcchem. 1968, 22, 214-224.
(18) Mahier, H. I?.In "Trace Elements": Lamb, c. A,, Bentley, 0.G,Beattie, J. M., Eds.; Academic Press: New York, 1958; p 311. (19) Fiereck, E. A. hl "Fundamentals Of ChliCd Chemistry"; Tietz, N. W., Ed.; W. B. Saunders: Philadelphia, 1970; p 956. (20) , . "The Bio-Science Laboratories Handbook". l o t h ed.: Bio-Science L&oratories: Van Nuys, CA, 1973; p 208. (21) Meii, L. D.; Maloy, J. T. Anal. Chem. 1975, 4 7 , 299-307. (22) Decker, L. A,, Ed. "Worthington Enzyme Manual"; Worthington Biochemical Corp.: Freehold, NJ, 1977; pp 58-60.
RECEIVED for review July 14, 1980. Accepted September 16,
411-417.
1980. This work has been supported by the National Science Foundation [Grant N ~ CHE-79239561 , and it has been presented in part during the 7th Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies [ S p p o s i u m On Andysis by Philadelphia, PA, Sept 30, 1980.
Determination of Vitamin C with Chloramine-T Krishna K. Verma' and Ani1 K. Guiati Department of Chemistry, University of Jabalpur, Jabalpur 48200 1, India
Vltamin C is determined In pharmaceutical preparations by titration with chloramine-T in the presence of starch-potasdum iodide or methyl red-potassium bromide. Iron(I1) does not Interfere. Sulfite and sulfhydryl compounds, which interfere severely, are masked by a prereaction with 2-furfuraldehyde and acrylonitrile, respectlveiy. Mixtures of vitamin C, sulfite, and glutathione or cysteine are analyzed.
Table I. Determination of Vitamin C in Pharmaceutical Preparations vitamin C, mg manufacturers NBS specifi- present titracation method" tiona Tablets 500 489 491 500 503 501 500 492 491 500 497 496
sample Chemical methods for the determination of vitamin C are based on the reducing properties of its 1,Zenediol group. A number of these methods have been reviewed (1). Oxidation with 2,6-dichlorophenolindophenolis the extensively used method; however, it is vitiated by the susceptibility of the dye to react with other reducing materials, e.g., cysteine and glutathione, which are frequently present in biological fluids (2), iron(I1) which is particularly found in food stuffs ( 3 , 4 ) , or sulfite commonly added as a preservative in soft drinks. Titration with N-bromosuccinimide has been reported to tolerate iron(I1) ( 5 , 6 ) ,albeit, the reagent is only poorly stable and reacts with cysteine, glutathione, and sulfite (7). Chloramine-T (CAT) is used in the present work as a stable titrant for determining vitamin C. Test solutions are titrated in the presence of either acidified potassium iodide and starch or acidified potassium bromide and methyl red. Iron(I1) does not interfere in either method but sulfhydryl substances and sulfite interfere severely. Treatment of a test solution with 2-furfuraldehyde or with acrylonitrile before titration has been found to mask sulfite and sulfhydryl compounds, respectively. Sulfite and sulfhydryls both can be rendered nonreducing by a prereaction with acrylonitrile. These reactions permitted the determination of vitamin C alone in the presence of these substances and the analysis of their mixtures.
EXPERIMENTAL SECTION Reagents. CAT,0.01 M solution, was prepared by dissolving 2.81 g of sodium N-chloro-4-toluenesulfonamide trihydrate in 1 L of water and standardized iodometrically (8). Sodium tetrathionate, 0.025 M solution, was made afresh by titrating 100 mL of 0.1 M sodium thiosulfate with 0.05 M iodine to the first appearance of iodine color which is bleached by dropwise addition of 0.01 M thiosulfate. This solution is purged with nitrogen and stored in a dark bottle. Phosphate buffer, pH 7,was prepared by dissolving 117.7 g of dipotassium hydrogen orthophosphate and 44.1g of potassium
Cecon (Abott) Celin (Glaxo) Suckcee (IDPL) Citravite (Pharmed) Ascorbic acid (Cadila) Sorvicin (East India) Chewcee (Lederle) Becosules (Pfizer) Cebexin (IDPL) Becadexamin (Glaxo) Calcium-Sandoz (Sandoz) Redoxon (Roche)
500
500
502
500
482
485
500
504
506
297
301
502 28
500 29
501
503
499
496
Capsules b 300 500 30
Injections 500 500
a Average of three determinations. Capsules have excipients, e.g., nicotinamide, thiamine hydrochloride, riboflavine, calcium pantothenate, pyridoxine hydrochloride, folic acid, cyanocobalamin, ferrous fumarate, etc. Injections have excipients, e.g., methyl- and propylparaben and calcium gluconogalactogluconate.
dihydrogen orthophosphate in 1 L of water. A 1% (v/v) aqueous solution of 2-furfuraldehyde and 0.01% indicator solution of methyl red in 95% ethanol were used. Acrylonitrile waa distilled before use. All other chemicals were of high purity. Standard solutions of the test compound were prepared by dissolving the right amounts of high purity materials in deionized water. Solutions of vi& C were standardized by titration with 2,6-dichlorophenolindophenol(9) of sodium sulfite with iodine
0003-2700/80/0352-2336$01.00/0 0 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table 11. Determination of Vitamin C in the Presence of Sulfite and Cysteine mixt no.
vitamin C amt added, mg C found,d taken,'" mg sulfiteb cysteineC mg 2.10 3.86 5.32 7.22 9.18
1
2 3
4 5 6
11.31
4.8f
2.06 3.88 5.29 7.26 9.09 11.38
8.1
2.5 4.9
3.4 5.6 4.4 2.6
CVe 0.5 0.6 0.9 0.8
1.2 1.0
Standardization with 2,6-dichlorophenolindophenol Standardization with iodine (10). StandardizaAverage of six detertion with o-iodosobenzoate ( 1 1). minations. e CV = coefficient of variation. f 2-Furfuraldehyde used for masking. a
(9).
~~~
Table 111. Determination of Mixtures of Vitamin C with Sulfite or Glutathione amt taken, mg mixt vitamin sulfite no. C(I) (11) 1
2 3 4 5 6 7 8
9 10 11
12
1.73 3.47 4.33 5.20 6.93 8.67 8.30 6.52 5.24 4.21 3.36 1.80
glutathione (1111
I (cv)'
11.06 14.89 19.26 22.55 24.38 30.86
1.75 (0.5) 3.50 (0.4) 4.37 (0.5) 5.26 (0.5) 6.85 (0.4) 8.73 (0.4) 8.42 (0.8) 6.61 (1.0) 5.30 (0.8) 4.40 (1.2) 3.43 (1.6) 1.85 (1.5)
8.8OC 7.05 4.3gc 5.28 3.52 1.76'
amt found," mg
II/III ( c v ) ~ 8.76 (0.6) 7.00 (0.5) 4.38 (0.5) 5.26 (0.5) 3.50 (0.6) 1.72 (0.8) 11.00 (0.8) 14.71 (0.9) 19.12 (1.0) 22.36 (1.1) 24.12 (1.3) 30.65 (1.4)
CV = coefficient Average of eight determinations. Sulfite masked with acrylonitrile; for the of variation. rest 2-furfuraldehyde was used. a
(IO) and of sulfhydryl substances with o-iodosobenzoate (11). Methods. Determination of Vitamin C i n Pharmaceutical Preparations. A finely ground tablet or the capsule itself is stirred with about 30 mL of deionized water containing 1mL of glacial acetic acid. After 15 min the residual solid is filtered off on Whatman No. 42 paper and washed with water. The filtrate is made up to a known volume. Injections are directly diluted. A suitable volume of solution is taken in a 100-mL Erlenmeyer flask, diluted to 20 mL, and mixed either with 0.5 g of potassium iodide, 1 mL of 0.5% starch, and 2 mL of 0.1 M sulfuric acid or with 0.5 g of potassium bromide, 2-3 drops of methyl red, and 2 mL of 1M sulfuric acid and titrated with 0.01 M CAT to the appearance of a blue color of starch-iodine or to the sharp bleaching of the red color of methyl red. Determination of Vitamin C i n the Presence of Sulfite. A known aliquot of test solution (about 10 mL) is treated with 5 mL of 2-furfuraldehyde and allowed to stand for about 15 min. The mixture is diluted to 20 mL and mixed with 0.5 g of potassium iodide, 1mL of 0.5% starch, and 2 mL of 0.1 M sulfuric acid. The
vitamin C is titrated with 0.01 M CAT to the appearance of a blue color. Determination of Vitamin C i n the Presence of Sulfhydryl Substances andlor Sulfite. A known aliquot of test solution (about 10 mL) is treated with 5 mL of phosphate buffer and 1 mL of acrylonitrile. The mixture is left for about 5 min when sulfite alone is present or for about 15 min when sulfhydryl substances are present with vitamin C. Thereafter, the contents are mixed with 5 mL of 0.1 M sulfuric acid, 0.5 g of potassium iodide, and 1 mL of 0.5% starch and titrated with 0.01 M CAT to a sharp blue color. Determination of Mixtures of Vitamin C with Sulfite or Glutathione. An aliquot of mixture is diluted to 25 mL and mixed with 0.5 g of potassium iodide and 1 mL of 0.5% starch. The contents are acidified with 2 mL of 0.1 M sulfuric acid and titrated immediately with 0.01 M CAT to a blue color. This gives a total of vitamin C and sulfite or glutathione. The vitamin C alone is determined by masking sulfite with 2-furfuraldehyde or acrylonitrile and glutathione with acrylonitrile as described before. Sulfite or glutathione is obtained by difference. Determination of a Mixture of Vitamin C with Cysteine and Sulfite. To determine a total of three components, an aliquot of mixture (about 10 mL) is mixed with 5 mL of phosphate buffer and 10 mL of tetrathionate. The contents are shaken for 1 min and then mixed with 0.5 g of potassium iodide, 1 mL of 0.5% starch, and 2 mL of 0.1 M sulfuric acid and titrated immediately with 0.01 M CAT to a blue color (titer I). An equal but second aliquot of mixture is treated with 5 mL of 2-furfuraldehyde for 15 min to tie sulfite; thereafter, the above method using tetrathionate is repeated to yield a totalof vitamin C and cysteine (titer 11). Sulfite is obtained by difference of titer I and 11. A third but equal aliquot of mixture is reacted with 1 mL of acrylonitrile in 5 mL of phosphate buffer for 15 min to tie cysteine and sulfite and thereafter mixed with 2 mL of 0.1 M sulfuric acid, 0.5 g of potassium iodide, and 1mL of 0.5% starch and titrated with 0.01 M CAT to a blue color to yield vitamin C alone (titer JII). Cysteine is obtained from the difference of titer I1 and 111.
RESULTS AND DISCUSSION CAT reacts with acidified iodide or bromide and yields iodine or bromine which reacts with vitamin C, and in both cases dehydroascorbic acid is formed. MeC6H4SO2NNaC1 2HI (or 2HBr) MeC6H4S02NH2+ NaCl + I2 (or Br,) (1)
+
-
The methyl red bleached end point is sharper than the appearance of starch-iodine blue color due to the slow rate of reaction between vitamin C and iodine at the equivalence point. Diluting the test solution with water averts fleeting blue end points. Substances which do not interfere with either indicator when present u p to 10-fold molar excess of vitamin C include iron(I1) sulfate, glucose, sucrose, maltose, lactose, starch, thiamine hydrochloride, citric acid, oxalic acid, tartaric acid, acetylsalicylic acid, maleic acid, phenacetin, alanine, serine, glycine, and urea. Some other examples are given in the footnote of Table 1. Sulfanilamide, isonicotinic acid hydrazide, methionine, tryptophan, biotin, and cystine when
Table IV. Determination of Mixtures of Vitamin C with Sulfite and Cysteine
'"
2337
amt taken, mg mixt no. vitamin C (I) sulfite (11) cysteine (111) I (CV)b 1 1.80 1.61 10.95 1.81 (0.6) 2 3.52 3.22 6.21 3.58 (0.5) 3 4.39 4.02 8.76 4.42 (0.8) 4 5.28 4.82 13.14 5.34 (0.8) 5 6.71 5.39 4.38 6.79 (0.7) 6 7.88 3.42 2.31 8.06 ( 0 . 5 ) Average of eight determinations. CV = coefficient of variation.
amt found," mg I1 (CV)'
I11 (CV) b
1.58 (0.5) 3.25 (0.5) 3.98 (0.5) 4.85 (0.4) 5.32 (0.6) 3.39 (0.6)
11.08 (1.0) 6.12 (0.8) 8.68 (0.6) 13.00 (1.0) 4.26 (0.5) 2.34 (0.5)
2338
Anal. Chem. 1980, 52,2338-2342
present in the aforesaid amounts do not interfere when iodide and starch are used but vitiate the results badly with methyl red and bromide even when present in traces. Amounts equal to that of vitamin C of 4-hydroxybenzoic acid, salicylic acid, N-acetyl-4-aminopheno1,and tyrosine produce about 1 % high results when methyl red and bromide are used but can be tolerated up to 20-fold molar excess of the determinant with the other indicator. Thiosulfate, sulfide, thiourea, and other strongly reducing species, e.g., hydrazine and hydroxylamine, interfere with both the indicators. Sulfite forms a usual complex with 2-furfuraldehyde or acrylonitrile by nucleophilic addition to activated T orbital of carbonyl or olefinic group yielding a nonreducing product. Formaldehyde reacts with vitamin C affecting its reducing properties and, therefore, cannot be employed to tie sulfite. Sulfhydryl substances undergo cyanoethylation with acrylonitrile via the same mechanism.
RSH
+ CH,=CHCN
-*
RSCHlCHzCN
(3)
T h e reducing thiol group is blocked. Alkali hydroxides and tetraalkylammonium hydroxides are powerful catalysts for cyanoethylation (12, 13) but in their presence vitamin C deteriorates. Phosphate buffer of p H 7 was found to catalyze the reaction, although less readily (2 min with hydroxides as compared to 15 min with phosphate buffer), but the vitamin C content is unaffected. Glutathione when present with vitamin C can be oxidized smoothly to its disulfide when potassium iodide and starch are used.
2GSH
+ I2
-
GSSG
is avoided in the present method by blocking the thiol group through cyanoethylation. Cysteine gives variable recoveries when determined under the condition used for glutathione. This is ostensibly due to the ready conversion of cysteine to a number of oxidation states (16). The tetrathionate method has been found to determine cysteine accurately, the reaction being
CySH + S40G2-
+ 3Br2 + 3H20
-
LITERATURE CITED Gupta, D.; Sharma, P. D.; Gupta, Y. K. Talanta 1975, 2 2 , 913. Ehsfcfd. R. E.: Huennekens, F. M. J. Am. Chem. SOC. 1955, 77, 3873. Basu, K. P.; Nath, M. C. J. Indian Chem. SOC. 1938, 15, 133. Penney, J. R.; Zilva, S. S. Biochem. J. 1945, 39, 392. Barakat, M. 2.;Abdel Wahab, M. F.; El-Sadr, M. M. Anal. Chem. 1955, 2 7 , 536. (6) Evered, D. F. Analyst (London) 1980, 8 5 , 515. (7) Verma, K. K.; Bose, S. Z. Anal. Chem. 1975, 274, 126. (8) Berka, A.; Vultarin, J.: Zyka, J. ”Newer Redox Titrants”; Pergamon: Oxford, 1965; p 37. (9) Franke, W. In “Modern Methods of Plant Analysis”; Paech, K., Tracey, M. V., Eds.; Springer-Verlag: Berlin, 1955; Vol. 11, p 95. (10) Vogel, A. I. “A Text-Book of Quantitative Inocganic Analysis”; Longman: London, 1978; p 383. (11) Verma, K. K.; Bose, S. Analyst (London) 1975, 700,366. (12) Misra, G. S.; Asthana, R. S. J. Pract. Chem. 1957, 4 , 270. (13) Wronski, M. Analysf (London) 1980, 8 5 , 526. (14) Verma, K. K. Curr. Sci. 1978, 4 7 , 82. (15) Kreshkov, A. P.; Oganesyann, L. B. Zh. Anal. Khlm. 1973, 2 8 , 2260. (16) Danehy, J. P.: Oester, M. Y. J. Org. Chem. 1987, 32. 1491.
(5)
A method based on these two reactions of different stoichiometry has been reported to analyze mixtures of vitamin C and glutathione or cysteine (15);however, inaccuracy results due to the difference between a small and a large titer. This
(6)
(1) (2) (3) (4) (5)
+ 2HI
GS03H + 6HBr
CySS203- + S203’-+ H”
The thiosulfate liberated in an amount equal to that of cysteine is in fact titrated so that measured amounts of tetrathionate need not be added provided there is a sufficient excess to complete the above reaction. In Table I results are given for the determination of vitamin C in pharmaceutical preparations and compared with those obtained by using N-bromosuccinimide (5).Quantitative data for the titration of vitamin C in the presence of thiol and sulfite are given in Table 11, whereas results for the analysis of their mixtures are presented in Tables I11 and IV.
(4) However, if potassium bromide and methyl red are employed, the oxidation goes further yielding a sulfonic acid ( 1 4 ) .
GSH
-
RECEIVED for review August 1, 1980. Accepted August 28, 1980.
Differential Thermal Lens Calorimetry N. J. Dovichi and J. M. Harris’ Department of Chemistry, University of Utah, Salt Lake City, Utah 84 112
A thermal lens calorimeter having a differential response is reported. The reference and unknown samples are located on opposlte sides of the waist of a slngle Gaussian laser beam. This arrangement produces a reference correction optically, by encoding the difference between the absorbance of two samples in the divergence of the beam.
The thermal lens effect, first reported by Gordon et al. ( I ) , has been successfullyapplied to the calorimetric measurement of minute absorptivities cm-’) of “transparent” materials (2-4), recording of absorption spectra of molecular transitions having low probability ( 5 , 6), and quantitative determination of trace level samples (7-9). Since the lower limit of detection of this technique is often dominated by the background absorbance of the sample matrix or the solvent, 0003-2700/80/0352-2338$0 1.OO/O
clearly analytical applications of the method would benefit by an experimental arrangement whereby the difference in absorbance between reference and unknown samples could be directly determined. In this work, we report a simple thermal lens calorimeter suitable for these applications. The differential response of the system arises from the antisymmetric dependence of the thermal lens effect on the position of the lens relative to a waist in a Gaussian beam. The theory of this response is developed by using the ray transfer matrix method and evaluated experimentally. The precision of this technique is compared with single thermal lens results for background limited samples.
THEORY In a thermal lens experiment, a lenslike element is formed within a sample from a radial temperature gradient caused 0 1980 American Chemical Society
