46
Anal. Chem. 1985, 57,46-51
Determination of Conjugated Glucuronic Acid by Combining Enzymatic Hydrolysis with Lucigenin Chemiluminescence Lori L. Klopf and Timothy A. Nieman* Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
Determlnatlons are carrled out In a contlnuous flow system. Samples pass through a column packed wlth controlled poroslty glass on whlch the enzyme P-glucuronldase ls Immoblllzed; glucuronides are hydrolyzed to glucuronlc acld and the corresponding aglycon. The effluent from this column combines wlth streams contalnlng NaOH, luclgenln (N,N’-dlmethyl-9,9’-dlacrldlnlum nttrate), and sodlum dodecyl sulfate. Chemllumlnescent emlsslon lntenslty Is proportional to the free glucuronic acld concentration and therefore to the orlglnal glucuronide concentration. Glucuronides tested were phenyl, nttrophenyl, methylumbelllferyl, bromonaphthyl, estradiol, and androsterone glucuronlde. Detectlon llmlts are 5-10 pM, and working curves are h e a r up to 2 mM. Preclslon Is 2% relative standard devlatlon. Other organic reductants, which may be present In blologlcal flulds and would Interfere In chemllumlnescent measurements, are ellmlnated from these samples by HPLC separatlon on an anlon exchange column located between the enzyme column and the addltlon of chemllumlnescent reagents. Urine samples are analyzed by this method and the results compared to a colorlmetrlc analysls.
Lucigenin (N,N’-dimethyl-9,9’-diacridinium nitrate) is known to undergo chemiluminescent (CL) reaction in aqueous alkaline solution in the presence of reductants and oxygen (1, 2). Work in this laboratory has made use of this CL reaction to develop methods for determination of clinically important organic reductants (3,4). Such reductants include ascorbic acid, creatinine, galactose, glucose, glucuronic acid, glutathione, lactose, and the uric acid. Heparin, a polymer of glucosamine and uronic acids, is similarly determined (5). In all these cases, the general CL advantages of wide working ranges and low detection limits are realized. The reductant of interest in this present work is glucuronic acid. The primary function of this sugar acid in the body is to combine with a variety of “foreign” molecules (e.g., drug metabolites or other potentially toxic organic species) to form conjugates called glucuronides. These glucuronides are more water soluble at physiological pHs than were their precursors; consequently they are easily eliminated from the body (6). Because most of these conjugation reactions occur in the liver, an accurate measure of the total amount of glucuronic acid in biological fluids would be a good indication of the proper functioning of the liver. In the past, the predominant technique used for the determination of free and conjugated glucuronic acid has been a colorimetric reaction with naphthoresorcinol that was developed by Fishman and Green (7); several modifications of this technique also have been reported (8-11). The major disadvantages of these techniques are (a) the long times necessary for the acid hydrolysis step and for the reaction with naphthoresorcinol, (b) the necessity of an extraction step for the removal of the pigment before a measurement can be made, and (c) the unsuitably of these techniques for automation in a clinical laboratory. 0003-2700/85/0357-0046$0 1.50/0
The use of the lucigenin CL reaction has been investigated in this work for the analytical determination of glucuronic acid in the form of glucuronides. One of the main limitations of many CL techniques is lack of selectivity toward particular analytes. This problem can be alleviated by coupling a sensitive but general CL reaction with a very specific enzymatic reaction. In this manner, analytes like glucose, cholesterol, uric acid, amino acids, and aldehydes produce their hydrogen peroxide equivalent via the appropriate oxidase enzyme, and the enzymatically generated peroxide is quantitated with luminol or peroxyoxalate CL (12). The enzyme 6-glucuronidase has been used in this study to specifically react with glucuronides to produce glucuronic acid and the corresponding organic compound (aglycon). The glucuronic acid will then react with an alkaline solution of lucigenin to produce CL. An example of this reaction is shown in Figure 1with phenyl P-D-glucuronide. The 6-glucuronidase has been immobilized and packed into a small column, described as an immobilized enzyme reactor (IMER). After passing through this reactor in a buffer a t pH 6.8, the glucuronide bond is hydrolyzed and the glucuronic acid concentration can be determined by the lucigenin CL reaction. In this paper, the lucigenin CL response in this continuous-flow system is studied for six different glucuronides, both individually and in mixtures. The biological fluids which would be tested for their total glucuronic acid concentration include urine and blood serum. These samples also contain other organic reductants which would certainly interfere in a determination of glucuronic acid by this CL method. Various techniques were tried here to eliminate these interferences, but the only successful one involved the combination of this technique with a chromatographic separation of glucuronic acid from the other reductants. Other reports have also been made of combining HPLC systems with immobilized enzyme reactors in the same flowing stream for additional selectivity before detection (13-16). In this report, the glucuronic acid concentration was determined in urine samples using the proposed CL technique and these values are compared with those from a colorimetric reaction with naphthoresorcinol.
EXPERIMENTAL SECTION Instrumentation, The glucuronide analyses were carried out on the continuous-flowsystem illustrated in Figure 2. A Rainin Rabbit peristaltic pump was used to pump each of three channels at a flow rate of 1.0 mL min-l. Glucuronidesamples were injected into the buffer stream with a Rheodyne type 50 Teflon rotary valve with a 500-wL sample loop; smaller loops can be used when the sample volumes available are limited. The sample then would be carried through the immobilized enzyme reactor (IMER),which is a 70 X 2.0 mm stainless steel column packed with a wet slurry of @-glucuronidaseimmobilized on controlled pore glass. The sample in the buffer stream then would combine in a cross-type mixing chamber with the other two flowing streams (containing NaOH, and a mixture of lucigenin and sodium dodecyl sulfate (SDS)) to produce CL. The system described above was used for the preliminary flow injection work with simple aqueous samples. For the application of this technique to biological samples, the continuous-flow system was changed slightly. The portion of 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 47 COOH
COOH
OH
OH
COOH
+
H J & HO
,
Lucigenin
+
02
base
CL
I
OH
Flgure 1. Glucuronide hydrolysis coupled with lucigenin chemiluminescence.
~ni;c+yo"- - 1-
I
- - -
1
Valve
Buffer
The HPLC detector used for the determination of the retention times of the various reductants was a differential refractometer (Waters Associates, MoPel R401). The equipment used in the colorimetric determination of glucuronic acid in urine samples included a Neslab waterbath (Model EX-200) for heating samples to a constant temperature and a Hewlett-Packard 8450A diode array spectrophotometer for making absorbance measurements. Reagents. The 0.4 mM lucigenin (Aldrich) solution used in the continuous-flowsystem also contained 0.4 mM SDS (Aldrich). Under these experimental conditions, this surfactant inhibita the precipitation of N-methylacridone, a product of this CL reaction, which would otherwise coat the components of the flow system and affect the measurement of the CL emission (18). A 0.6 M NaOH (Mallinckrodt) solution was used in another channel of the system, and the buffer used in the third channel during the initial experiments was 0.1 M phosphate buffer prepared from NaHzP04-Hz0(Baker) and adjusted to pH 6.80 (iO.01) with NaOH. (It was necessary to use sodium salts rather than potassium salts for the base and buffer reagents, since a displacement of the counterion in SDS would result in the precipitation of this surfactant as potassium dodecyl sulfate.) The buffer used with the HPLC system for separation of the reductants was 0.1 M acetate buffer prepared from sodium acetate (Mallinckrodt) and adjusted to pH 6.00 (iO.01) with 0.1 M acetic acid (Mallinckrodt). Glucuronic acid (sodium salt) and the following six glucuronides used in this study were all from Sigma: androsterone glucuronide, 6-bromo-2-naphthyl @-D-glucuronide,0-estradiol 17(@-D-glucuronide) (sodium salt), 4-methylumbelliferyl 0-D-glucuronide, p-nitrophenyl @-D-glucuronide,and phenyl 0-D-glucuronide. The corresponding aglycons used in studies of their effects on CL were androsterone, 6-bromo-2-naphtho1, @estradiol, and 4-methylumbelliferone (all from Sigma), p-nitrophenol (Aldrich), and phenol (Mallinckrodt). Other reductants used in interference studies for real samples included creatinine (ICN Pharmaceuticals), uric acid (Eastman Kodak), D-glucose (Mallinckrodt), dehydroascorbic acid (Pfaltz and Bauer), and glutathione (both the reduced and oxidized forms) and ascorbic acid (all from Sigma). The enzyme used in this system was Type IX @-glucuronidase (EC 3.2.1.31) (Sigma) derived frov E . coli, which is reported to be the preferred enzyme for the hydrolysis of certain steroids such as androsterone (19)and most estrogens (20). Other reagents used in the enzyme immobilization include sodium acetate (Mallinckrodt) and acetic acid (Mallinckrodt) for an acetate buffer (pH LO), sodium pyrophosphate (Fisher) and hydrochloric acid (Mallinckrodt) for a pyrophosphate buffer (pH 8.5), (3-aminopropy1)methyldiethoxysilane(Petrarch Systems), glutaraldehyde (Eastman Kodak), NaCl (Mallincrodt), and methanol (MCB Reagents). Controlled porosity glas (CPG) used fbr the immobilization was obtained from ElectroNucleonics with 37-74 pm diameter (200-400 mesh) and 547-& average pore size. Reagents used in the colorimetric analysis urine samples included hyrochloric acid (Mallinckrodt), naphthoresorcinol (Aldrich), and ethyl ether (Burdick and Jackson Laboratories, Inc.). Water from a Millipore Contiliental water pclrification system was used in the preparation of all solutions described above. Immobilization of @-Glucuronidase. The immobilization method used was based on the irocedure developed by Bowers and Johnson (21). The solid support, CPG, was first washed with nitric acid and then silanized by reacting for 2 h at 90 "C with (3-aminopropyl)methyldiethoxysilanein 0.05 M acetate buffer (pH 5.0). A solution of glutaraldehyde (12%) in 0.1 M pyrophosphate buffer (pH 8.5) was reacted with the Bctivated CPG for 2 h at room temperature to generate aldehyde functional groups on the CPG for the reaction with the enzyme. Excess glutaraldehyde was removed by rinsing with water and with the pyrophosphate buffer. The @-glucuronidase(21 IU/g of CPG) was attached by reaction overnight at 5 "C in the pyrophosphate buffer. The CPG was then washed with water, 1.0 M NaC1, water, and pyrophosphate buffer to remove any adsorbed or entrapped enzyme. (All water rinses wefe with Millipore-purified water.) The immobilized @-glucuronidasewas stored in 0.1 M phosphate buffer (pH 6.8) at 5 "C. Determination of IMER Adtivity. After the immobilized @-glucuronidasewas packed into a column, it was necessary to determine the quantity of glucuronide which could be hydrolyzed
NaoHL I
Lucigenin
t
Signal
Figure + SDS2. Flow system schematic:
6
BUFFER
PUMP
S
C
D
E I F
to
woste-g?f-+ to C L mixer
Flgure 3. Additional flow system schematic when using the HPLC system. Components shown include injection valve (A), guard column (B), IMER (C), column prefllter (D), analytical column (E), and four-way valve (F). Figure 2 enclosed by a dashed line was replaced by the HPLC system depicted in Figure 3. In this revised system the buffer is pumped at a 1.0 mL min-' flow rate by an Altex Model llOA HPLC pump. It first passes through a high-pressure injection valve (A) (Altex Model 210) with a 100-pL sample loop and an attached event marker (Altex Model 210-14) and then through a guard colum (B) (a 70 X 2.0 mm stainless steel column packed with Whatman Pellicular Anion Exchanger) and the IMER (C). Because some of the aglycons formed during the hydrolysis of glucuronides in the IMER are not soluble in aqueous solutions, a high-pressure column prefilter (D) (Scientific Systems) with replaceable 0.5-wm frits was located in front of the analytical column (E) to prevent clogging of the inlet frit of this column. The analytical anion exchange column used for separation of glucuronic acid from the other reductants in biological samples was a 250 mm X 4.6 mm column packed with Whatman Partisil SAX packing (10 pm). Both the effluent from this column and another buffer stream from the peristaltic pump entered a Rheodyne type 50 Teflon rotary four-way valve (F),which was installed on a pneumatic actuator (Rheodyne Model 5001) for automatic switching of the valve to interchange the two flowing streams. In this system only the portion of the HPLC column effluent containing glucuronic acid was allowed to pass through the valve to the CL mixing chamber; the rest of the sample was directed to the waste container. In both experimental situations described above, the reaction mixture from the mixing chamber was delayed for 1.0 min in a coil of Teflon tubing before passing through an in-house designed flow cell (17) with a 240-pL volume. The CL emission was detected by a photomultiplier tube (Hamamatsu R928 biased at 900 V) and the signal was amplified by a Pacific Precision Instruments Model 126 photometer, using a 3-s time constant filter to reduce the noise level on the CL signals. -These signals were recorded as a function of time on a strip chart recorder.
48
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
@ & H0"
HO
I
H
(01
.-VI
(cl
(bl
0 OH
C
a
c
C
D
O
H
\
/
Br
NO2
(dl
u
OO
0,4
0.8
1.2
p heny I -p-D-g lucuronide (mM)
Figure 4. Working curve for phenyl @-glucuronide.
as it made a single pass through the reactor. A known amount of p-nitrophenyl glucuronide was passed through the IMER in a flowing stream of 0.1 M phosphate buffer (pH 6.8), the hydrolyzed glucuronide was collected in a volumetric flask, and the amount of p-nitrophenol in the final solution was quantitated by measuring the absorbance of the solution at 400 nm. The hydrolysis efficiency of the IMER was initially 100% for a 500-pL sample of 0.5 mM p-nitrophenyl glucuronide and a 1.0 mL m i d flow rate. This hydrolysis efficiency was measured every 2-3 days for the first 6 months after the IMER was packed, and it was seen that after about 12 weeks the efficiency had gradually decreased to 91% hydrolysis in a single pass at 1 mL min-l. The efficiency of this IMER remained at 91% for at least four additional months. During the first few weeks of use, the hydrolysis efficiency could be maintained at a higher level by alternating the direction of flow through the IMER every few days. A new batch of enzyme was immobilized for the experiments dealing with applications of this technique to biological samples. The hydrolysis efficiency of the IMER packed with this enzyme remained at 100% for all of the subsequent experiments done in the work with interferences and application to analysis of urine samples. Procedure for Colorimetric Determination of Glucuronic Acid in Urine Samples. The values for total glucuronic acid concentration determined in urine samples by the proposed CL method were compared to an analysis of the same samples done by Cornillot's adaptation of the naphthoresorcinol colorimetric method (8). In this analysis, 2.0 mL of each urine sample was diluted to 25.0 mL with water. Then 2.0 mL of each diluted sample was combined with 4.0 mL of 3 M HCl in a loosely stoppered-test tube, which was then heated for 30 min in a 100 "C water bath to hydrolyze the glucuronides. After the sample was cooled to room temperature, 1.5 mL of each sample was combined with 0.5 mL of a 0.6% naphthoresorcinol solution. (This reagent was prepared by boiling 0.6 g in 100 mL of water for 3 min, cooling to room temperature, filtering into a dark glass bottle, and storing at 5 "C. Because of the instability of this reagent, it was used within 48 h of preparation.) These stoppered solutions were heated in a sandbath for 60 min at 100 "C. After the solutions cooled in ice, 5.0 mL of ethyl ether was added to each sample to extract the pigment for the absorbance measurements (A, = 570 pm). Glucuronic acid standards were also prepared as described above with the omission of the first heating step.
RESULTS AND DISCUSSION Results with Glucuronic Acid. For this coupled enzyme-lucigenin CL technique to be successful for assay of glucuronide samples, the observed CL emission intensity must be linear with respect to the concentration of glucuronic acid. A CL working curve for glucuronic acid is linear from 8 pM to 2 mM. The linear regression data for triplicate measurements at each of nine concentrations over this range are slope 50.2 f 0.5 pA M-l, y intercept -0.54 f 0.34 nA, and correlation coefficient 0.9997. Precision for individual solutions is 2 %
CH3
(e)
(fl
Flgure 5. Glucuronides tested: (a) phenol, (b) androsterone, (c) estradiol, (d) nitrophenol, (e) 6-brom0-2-naphtho1, (f)' 4-methylumbelliferone.
Table I. CL Intensities of Glucuronides Relative to Glucuronic Acid glucuronide D-glucuronic acid 4-methylumbelliferyl P-D-glucuronide p-nitrophenyl 0-D-glucuronide 6-bromo-2-naphthyl P-D-glucuronide phenyl 0-D-glucuronide P-estradiol 17(P-D-glucuronide) androsterone glucuronide
re1 CL intens 1.00
0.96 0.94
0.86 0.79 0.69 0.11
relative standard deviation or better. The detection limit for glucuronic acid is 5 pM or 1 mg L-l. The detection limit is a decade lower than that reported earlier for lucigenin CL using stopped flow instrumentation (3). This working range is satisfactory for the determination of glucuronides in biological fluids, since the normal adult concentration of conjugated glucuronic acid is in the range of 0.8-2.4 mM in urine and 0.10-0.23 mM in blood (22). Results with Glucuronides. Most initial work was done with phenyl glucuronide; its chemical reactions in this system were described earlier in the discussion for Figure 1. A working curve for this glucuronide is shown in Figure 4 for a concentration range of 8 pg to 1 mM. This plot of CL intensity vs. concentration of phenyl glucuronide is seen to be quite linear; the inset in this figure indicates the linearity of the working curve even a t low concentrations. Other molecules conjugated to glucuronic acid were then obtained for analysis with this system; all compounds tested are shown in Figure 5. In each case, glucuronic acid is conjugated at the hydroxyl group (the C17hydroxyl for p-estradiol). These glucuronides were chosen because of their different molecular sizes and chemical groups present, to see if these factors would affect either the enzymatic hydrolysis or the subsequent CL reaction. Working curves were made for each of these glucuronides over the same concentration range as for phenyl glucuronide. Each was linear, with a slope similar to that in the plot for phenyl glucuronide in Figure 4. The curve for androsterone glucuronide had a much shallower slope, however; it is only one-tenth that of a glucuronic acid working curve made under the same conditions. The poor response for this glucuronide is caused by a slower rate of hydrolysis for this particular substrate. Androsterone glucuronide is a 3-hydroxy-5acompound, which has been found in previous studies to be one of the most slowly hydrolyzed steroid conjugates (19). The relative signals for 0.4 mM solutions of each glucuronide are compared to the signal for glucuronic acid in Table I. Methylumbelliferyl and nitrophenyl glucuronides yield signals essentally identical with that for glucuronic acid. The aglycons of all six of these glucuronides (4-methylumbelliferone, p -
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
Table 11. Results for Mixtures of Glucuronides sample
CL intens, nA
mixture of 6 (at 0.1 mM each) [glucuronic acid] = 0.6 mM
25.6" 31.6
mixture of 5 (at 0.1 mM each) [glucuronic acid] = 0.5 mM
26.0" 26.1
"These values corrected for the 91% IMER hydrolysis. nitrophenol, 6-bromo-2-naphtho1, phenol, @-estradiol,and androsterone) were tested to see if they had any effect on the CL reaction with glucuronic acid. A slight suppression in the CL intensity was noted only in the presence of relatively high concentrations of phenol. Therefore, the lower CL signals seen €or the remaining three glucuronides are due to incomplete hydrolysis of these substrates at this concentration as they pass through the IMER at a 1.0 mL mi& flow rate. The glucuronide content in samples of biological fluids would actually consist of a combination of many different glucuronides. A mixture was prepared for analysis which contained 0.1 mM of each of the six glucuronides used (Figure 5). Because of the low results obtained earlier for the acdrosterone glucuronide, another mixture was prepared which contained 0.1 mM of each of the other five glucuronides. These two mixtures were assayed by this technique and the results compared to those from the corresponding concentrations of glucuronic acid samples. It can be seen in Table I1 that although the signal for the first mixture is slightly low (as expected due to the androsterone glucuronide), very good agreement is obtained for the second mixture. In this experiment it is important to note that the hydrolysis of each glucuronide appears to have been complete, and the CL signal was not suppressed by the presence of the phenol aglycon; this is probably due to the lower (and more realistic) concentration of each of these species. Application. This lucigenin CL system coupled with enzymatic hydrolysis is superior to previously used methods for determination of conjugated glucuronic acid in terms of both the simplicity and the speed of the analysis. Both the working range and the detection limit are satisfactory for assay in biological fluids such as urine and blood. Work has been done in this laboratory on approaches to eliminate the interference from other organic reductants normally found in such samples. These reductants inclade ascorbic acid, creatinine, galactose, glucose, lactose, uric acid, and glutathione (this compound is not present in urine). The methods which have been evaluated for this analysis include a Somogyi-Nelson sample pretreatment, kinetic differentiation, bulk electrolysis, and HPLC separation. The Somogyi-Nelson sample pretreatment (23,H)involved removal of several of the possible interferents by cationic protein precipitation from the addition of Ba(OH), and ZnS04. Although most of the interferences were removed from simulated biological samples by this technique, it was found to be unsatisfactory since most of the free glucuronic acid and a small portion of the glucuronidespresent were also removed with the precipitate. This technique could only be used for an estimate (f30%) of the concentration of conjugated glucuronic acid in a sample. The kinetic differentiation method was based on the fact that for general enzyme-catalyzed reactions in which the substrate concentration is below its Michaelis-Menten constant, the initial rate of the conversion of substrate to product is proportional to the substrate concentration (25). By leaving a glucuronide sample in a low-activity IMER for increasing amounts of time before it reacts with the CL reagents, the extent of the hydrolysis reaction will increase in proportion to the time spent in the IMER. A plot of CL intensity vs.
49
sample time spent in the IMER yields a slope which is proportional to the initial rate of the enzymatic hydrolysis and, therefore, can be directly correlated with the concentration of the conjugated glucuronic acid in a sample. Interferences present in a sample may raise or lower the CL intensity, but they would have no effect, on the slope of the plot described above as long as no species were present to activate or inhibit the enzyme, Because the magnitude of the change in slope with glucuronide concentration was relatively small but the intensity of the CL signal could be greatly increased with the presence of interfering reductants, significant problems were observed in obtaining accurate results for the glucuronic acid concentration. A third method used to try to eliminate interferences from reductants present in biological samples involved an on-line bulk electrolysis of the sample as it passed through the continuous-flow system. Although it was hoped that the products formed by this electrolysis would not affect the intensity of the CL signal, it was found that some of these newly formed species still enhanced the CL reaction of lucigenin with base. Dehydroascorbic acid, the oxidation product of one of the major interferences, ascorbic acid, was actually found to produce a greater CL intensity per mole than the ascorbic acid. Therefore, this oxidation by electrolysis was not suitable for elimination of interferences. The method which was found to be successful was an HPLC system in which the glucuronic acid (formed in the IMER) is separated from the other interfering reductants. Because of the presence of the IMER in this chromatographic system, the mobile phase chosen for the separation had to meet certain requirements: (a) it could contain little or no nonaqueous solvents, and (b) it had to be a buffered system near pH 6.8, which is the optimum pH for the activity of @-glucuronidase. Separations were tried with both phosphate and acetate buffers adjusted to various pHs, using three different anion exchange columns, one aminopropyl column in the anion exchange mode, and one reversed-phase C-18 column. (These columns included a Vydac anion exchange column (500 X 4.6 mm), a Whatman Partisil SAX column (250 X 4.6 mm), a Wescan Anion column (250 X 4.6 mm), a Zorbax NH2 column (250 X 4.6 mm), and a Rainin Microsorb C-18 column (100 X 4.6 mm).) The best separation was obtained with the Partisil SAX column using a 0.1 M acetate buffer at pH 6.00 for the mobile phase; the chromatograms of the individual species of interest are superimposed on the same time scale in Figure 6. (A chromatogram of dehydroascorbic acid is included in the figure because ascorbic acid in real samples is partially converted to this product by air oxidation. The retention time of the oxidized form of glutathione is 19.1 min, so this chromatogram was not included.) As seen in Figure 6, the glucuronic acid peak is separated sufficiently from the other peaks to eliminate interference problems. A diagram of the changes made to the original flow system to include this HPLC system is shown in Figure 3. The four-way valve was triggered by an event marker on the injection valve and automatically set, using an electronic circuit coupled with air-driven solenoid valves, to divert the HPLC effluent to the CL detection cell from 6.75 min to 8.58 min after injection of the sample. (This portion of the chromatogram in Figure 6 is indicated by dashed lines.) Except for this small part of the column effluent, all of the rest of the sample (containing any interfering reductants or aglycons) is sent to a waste container. Therefore, ths technique should be able to be used for the determination of the total glucuronic acid concentration in both urine and blood samples. CL was found to be superior for the detection of glucuronic acid compared to other detection techniques often used with HPLC systems. Glucuronic acid cannot be detected by ab-
50
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
Table 111. Total Glucuronic Acid Concentrations Determined in Urine Samples by Two Methods lglucuronic acid]. mM sample no.
CL method
colorimetric method
1 2 3 4
1.59
2.16 2.03 3.62
5 6 7 8
0
4
6
12
Time ( m i d
Figure 6. Refractive index chromatograms of glucuronic acid and
possible interferences: (a) glucose, galactose, lactose, or other unretained species, (b) dehydroascorbic acid, (c)creatinine, (d) glutathione (reduced form), (e) glucuronic acld, (f) ascorbic acid, and (9) urlc acid. sorption in the ultraviolet and visible regions or by fluorescence, because like other carbohydrates, it contains neither chromophores or fluorophores. Since the oxidation potential of glucuronic acid is greater than +1.20 v vs. Ag/AgCl, an electrochemical detector used with this system had a very high background current and was also very sensitive to pump noise. The combination of these factors made electrochemical methods too insensitive for detection of levels of glucuronic acid which would be found in biological systems. The refractive index detector was the best of the methods mentioned so far for the detection of glucuronic acid, but its detection limit by this method is only about 0.4 mM. Poor detection limits for carbohydrates by refractivity have also been noted by other authors (26,27). Since the range of glucuronic acid in urine is usually 0.8 mM to 2.4 mM and in blood is usually 0.10 mM to 0.23 mM, this method is not sensitive or selective enough to obtain accurate values in these biological fluids. The results obtained for a working curve for p-nitrophenyl glucuronide in this HPLC system using CL detection gave a linear plot with a detection limit of 10-15 pM,which is low enough for the analysis of glucuronic acid in either urine or blood serum. Interference studies with ascorbic acid, creatinine, and uric acid showed no effect on the CL signal using this chromatographic system. This technique was then tested with eight urine samples from healthy adults (five male and three female) between 24 and 35 years of age. These samples were collected from the first morning urination of each of the eight subjects, and were analyzed within 12 h of collection. The urine samples were each diluted by a 15 ratio with buffer to yield a final concentration of 0.1 M acetate buffer (pH 6.00). To prevent any particulates in the samples from entering the HPLC system, these samples were filtered first with a No. 42 (2.5-pm) filter and then with a 0.20-pm filter in a 13 mm diameter filter holder (Gelman Sciences, Inc.) as they were injected into the sample loop. (New filters were used for each urine sample.) The results for this determination of the total glucuronic acid concentration in each sample are listed in Table I11 along with the values obtained for the same samples by a naphthoresorcinol method. (The procedure for this colorimetric method is found in the Experimental Section.) As seen in Table 111, the glucuronic acid concentrations determined by the CL method proposed here are all somewhat
1.14 2.32 0.47 3.09 2.28 0.41 1.72
1.85 4.53 2.88 1.47 3.27
lower than the concentrations found by the naphthoresorcinol method used. In the article by Cornillot describing this naphthoresorcinol method (8),it was found that he had observed higher glucuronic acid levels in urine samples analyzed by his technique compared with other variations of this colorimetric method. Although he felt that the reason for these higher values was a complete hydrolysis of the glucuronides with a minimum of destruction of glucuronic acid by the harsh acid conditions, we believe that his high values compared to the CL method may be due to the effects of other species on the naphthoresorcinol reagent. While Cornillot did not mention any studies with interferences, other reports have been made of the positive reactions of ascorbic acid, creatinine, glucose, and other sugars with the naphthoresorcinol reagent (9,28). Further tests were done here to assess the interference of typical urinary levels of glucose, ascorbic acid, creatinine, and uric acid on the signal obtained for a sample containing glucuronic acid. While the addition of glucose (at the low levels found in urine) had no significant effect on the naphthoresorcinol absorbance at 570 nm, the addition of both ascorbic acid and creatinine gave very large positive errors in the absorbance value. Uric acid, however, was observed to have a noticeable negative effect on the naphthoresorcinol absorbance due to glucuronic acid. The precision of measurements made with this colorimetric method was also found to be much worse than those obtained with the proposed CL method: 33% RSD and 2% RSb, respectively. Since the CL method proposed here provides precise measurements and specifically eliminates interferences that might cause problems in the analysis, i t should be a much more reliable and accurate technique for the quantitation of total glucuronic acid in urine and blood samples than any of those previously reported. This CL system also could easily be automated, which would then require only a minimum amount of technician time for the analysis of a large number of clinical samples. Work is under way in this laboratory to develop this CL method for the on-line differentiation of free and conjugated glucuronic acid in biological samples. Further extensions of this technique may involve an HPLC separation prior to the IMER so that levels of individual glucuronides can be quantitated.
ACKNOWLEDGMENT The authors wish to thank P. R. Johnson of the University of Minnesota for helpful discussion concerning the enzyme immobilization procedure, W. H. Pirkle and K. Harlow of the University of Illinois for the loan of some HPLC columns, and R. M. Coates of the University of Illinois for the loan of a refractive index detector. Registry No. Lucigenin, 2315-97-1;p-glucuronidase, 9001-45-0; androsterone glucuronide, 1852-43-3;6-bromo-2-naphthyl 0-Dglucuronide, 22720-35-0; @-estradiol17-(P-~-glucuronide), 18066160-80-1;p-nitro98-0; 4-methylumbelliferyl-~-~-glucuronide, phenyl-P-D-glucuronide, 10344-94-2; phenyl-P-D-glucuronide, 17685-05-1.
Anal. Chem. 1985, 57,51-55
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51
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RECEIVED for review March 19,1984. Resubmitted September 24, 1984. Accepted September 24, 1984. This work was supported by the National Science Foundation (CHE-8106616). L.L.K. is grateful for fellowship support from the Phillips Petroleum Co.
Luminescence Quantum Counter Comparator and Calibration of New Quantum Counters for Light Intensity Measurements J. N. Demas,* T. D. L. Pearson, and Edward J. Cetron Chemistry Department, Chemistry Building, University of Virginia, Charlottesville, Virginia 22901
A new computerlred quantum counter (QC) comparator was developed. The comparator was designed to mlnlmlze systematic errors and exhlblts high preclslon and accuracy (
