Determination of Kow values for a series of aryl glucuronides

Full text of DOI:10.1007/s001289900955

Bull. Environ. Contam. Toxicol. (1999) 63:109-116
© 1999 Springer-Verlag New York Inc.
Determination of Kow Values for a Series of
Aryl Glucuronides
D. W. Kuehl, J. C hristensen
Mid-Continent Ecology Division, National Health and Environmental Effects Research
Laboratory, United States Environmental Protection Agency, Duluth, MN 55804, USA
Received: 16 February 1999/Accepted: 22 April 1999
Polynuclear aromatic hydrocarbons (PAHs) are a class of hazardous contaminants in
the aquatic environment that readily accumulate in animals. We have recently
become interested in understanding the formation, distribution, and elimination of
phase II metabolites of PAHs in fish (McKim et al. 1993) in support of the U.S.
EPA’s hazardous chemical risk assessment programs. Glucuronides are one of the
important phase II metabolites and are formed by the conjugation of glucuronic acid
with phase I metabolites of PAHs, hydroxylated PAHs (Clarke et al. 1991). The
commercial availability of aryl glucuronides for study is, however, limited. We have,
therefore, prepared a series of para substituted phenyl glucuronides, and wish to
report a simple yet effective sample cleanup procedure for their isolation from
microsomal incubation solutions, and features of ultra violet (UV) and electrospray
ionization mass spectrometric (ESI/MS) data for their structural characterization.
An important parameter in toxicokinetic modeling is the octanol/water partition
coefficient (Kow). This parameter has often been used to predict the accumulation
of contaminants from water to fish (Klamer and Beekman 1995); however, few Kow
values are available for modeling the behavior of phase II metabolites within an
animal. Therefore, Kow values for the synthesized glucuronides, along with a few
commercially available glucuronides, were determined using reversed-phase high
performance liquid chromatography (RP-HPLC). The measured values were
compared to those predicted by a substituent additive model, CLOGP (Leo and
Weinninger 1988). An assessment of this data is presented.
METHODS AND MATERIALS
Phenol, uracil, p-aminophenol, hydroquinone, p-cresol, b -naphthol, p-fluorophenol,
p-chlorophenol, p-methoxyphenol, p-t-butylphenol, and p-phenylphenol, were
obtained from Chem Services (West Chester, PA). Phenyl glucuronide , p-
nitrophenyl glucuronide,
b-naphthyl glucuronide, 6-bromo-2-naphthyl glucuronide,
trichloroacetic acid (TCA), formic acid, glycine, ammonium acetate, uridine 5-
diphosphoglucuronic acid (UDPGA) and BSA were obtained from Sigma-Aldrich
Chemical Co. (St. Louis, MO). Organic solvents were obtained from Fisher
All reagents were of the highest purity available.
Scientific (Fair Lawn, NJ).
Correspondence to: D. W. Kuehl
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Rainbow trout (Onchorynchus mykiss) liver microsomes were prepared and
characterized as previously described (Dady et al. 1991). Aryl glucuronides were
prepared and purified in the following manner. Trout liver microsomes (500 µL, 8-10
mg protein), MgCl2 (100µL of 100mM), UDPGA (100µL of 100mM), and a
substituted phenol (100µL of 100mM in Tris-HCl (100mM, pH 7.6)) were combined,
diluted to 1.5 mL with Tris-HCl (100mM, pH 7.6) and incubated with gentle mixing
for 24 hr at 25° C. Protein was removed from the incubation reactions by the addition
of either TCA (200µL, 0.6M containing glycine (0.3M)) or ice-cold ethanol (1 vol)
followed by centrifugation (10min, 10,00Xg). The supernatant was transferred to
a prewashed Quaternary Amine anion exchange (3mL) and/or a C18 Reversed Phase
column (3mL) (J.T. Baker, Phillipsburg, NJ), and eluted with 0.1% formic acid in
water (1-3mL) followed by 0.1% formic acid in methanol/water (5-50%, v:v, 1-
6mL). One mL fractions were collected.
Glucuronide characterization was done using a Nouveau Gold HPLC system fitted
with anUltrasphere C18 5µ RP-HPLC column (2X250mm) and a photodiode array
UV detector (Beckman Instruments, Fullerton, CA). The eluant was either A:1%
methanol, B:91% methanol or A:1% acetonitrile, B:51% acetonitrile, which were
buffered at 0.5, 1.0, 2.5, 5.0, or 20.0 mM sodium phosphate (pH 6.8-7.0,1:1, v:v,
monobasic:dibasic). The elution gradient was 100%A to 100%B in 15, 30, 60, 90,
or 120 min. Solvent flow was 120µL/min, at 22-23°C. Glucuronide standards were
1mM. The injection volume was 20µL. For mass spectrometric analyses, the HPLC
was connected to a Finnigan-MAT (San Jose, CA) TSQ-700 triple quadrupole mass
spectrometer fitted with an ESI source (3.5 KV). The mobile phase contained 0.1%
formic acid and 1.0mM ammonium acetate. MS/MS product ion spectra were
generated using 1torr Argon, with a collision energy of 25V.
Calibration of the HPLC column for Kow determinations was done by regressing the
Log of the shake flask Kow value (Hansch and Leo 1979) with the Log of the
measured HPLC capacity factor for each of six calibration standards, uracil, p-
aminophenol, hydroquinone, phenol, p-cresol, and b -naphthol. Phenyl phosphate
was used to establish column void volume. Phenyl phosphate and p-cresol were
added to each glucuronide solution as HPLC elution markers. Calculated Kow values
were obtained using CLOGP (Leo and Weinninger 1988).
RESULTS AND DISCUSSION
Methods for the synthesis and characterization of the para-substituted phenyl
glucuronides were developed using phenol to produce phenyl glucuronide. After
protein precipitation, a typical average yield was 28.5% (n=5). The SPE column
cleanup procedures resulted in very little product loss. The anion exchange column
was found to be very effective for the separation of glucuronides with low Kow
values from UDPGA, and the C18 reversed-phase SPE column effectively separated
glucuronides with higher Kow values from their starting phenol. An example
chromatogram, ES/MS spectrum, and UV spectrum for p-chlorophenyl glucuronide
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are shown in Figure 1. The photodiode array UV spectra for each glucuronide
showed two adsorption peaks, 220±5 and 270±5nm. The adsorption at 220nm was
generally stronger; however, 270nm often has less interferences and thus may be
more useful for detecting phenolic glucuronides. When ammonium acetate (1.0 mM)
was added to the HPLC mobile phase for the ESI/MS analyses, each compound gave
an intense ion at (M+18)+ (ammonium ion adduct). Liberato et al. (1983) reported
that the thermospray ionization spectrum of glucuronides showed an ion for the
ammoniated glucuronic acid fragment at m/z 194. This ion was also observed in the
ESI/MS/MS product ion spectra of phenyl-, p-nitrophenyl-, and naphthyl
glucuronides (data not shown). Precursor ion scanning using m/z 194 may then be
an very useful technique for the characterization of unknown glucuronides.
An excellent review of the theory of RP-HPLC for the determination of Kow values
He describes the role of both the mobile and
was presented by Braumann (1986).
stationary phases in the retention of analytes, and presents correlations between
HPLC capacity factors and Kow. Further he presents a discussion of the concept of
Kw, the Kow value obtained by extrapolation of Kow values obtained using isocratic
chromatography at various high percentages of organic modifier in the mobile phases
to an expected value at 100% water. This concept, along with additional discussions
of the influence of pH and ion-paring on Kow have more recently been reviewed
(Hsieh and Dorsey 1993, Lambert 1993 and Valko et al. 1993). In 1997, Finizio et
al. (1997) presented a review of the application of RP-HPLC to Kow determinations
with respect to OECD guidelines. OECD recommends (Klein et al. 1988), among
other things, 1. use of calibration standards with well established Kow values, 2.
calibration with at least six standards, and 3.elution with at least 25% water. All of
these reviews and recommended guidelines have generally been for assessing
In this
compounds of a wide variety of chemical classes with high Kow values.
study, however, we focus on only one class of compounds, highly water soluble
phase II metabolites. Analytical procedures, therefore, were less well established and
required some development.
The criteria for selection of column calibration compounds was that they should be
similar to the glucuronides, i.e. highly water soluble, at least partially aromatic, and
weakly acidic. The series p-aminophenol, hydroquinone, phenol, p-cresol, and b -
naphthol, which have Log Kow values of 0.04, 0.55, 1.48, 1.96. and 2.71 (averaged
values, Hansch and Leo 1979) respectively, was selected. Uracil was also selected
The HPLC mobile phases were buffered
because it has a low Log Kow value, - 1.05.
at neutral pH (1mM sodium phosphate, pH 6.8-7.0) to simulate the pH of living cells
or blood. The OECD recommends that the analyses be done at a pH two log units
below the pKa of the analyte (Klein et al. 1988). CLOGP estimates the pKa for all
of the glucuronides to be about 3.0. Chromatography two log units lower (pH 1.0)
is undesirable because, 1. it is not the pH of living organisms, 2. very few HPLC
columns can survive extended exposure to such low pH values, and 3. glucuronides
in solutions with a pH much greater than or less than neutral may hydrolyze.
Methanol was chosen as the mobile phase organic modifier because it has been most
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frequently used by other researchers reporting Kow values, and it is the most “water
like” organic solvent (Lambert 1993). A linear gradient rather than an isocratic
elution was chosen because analysis times could be shortened, and gradient analyses
have been shown to predict Kow with a precision of better than 0.5 Log units
(Klamer and Beekman 1995). It was important, however, to establish appropriate
gradient conditions. To do this, the percent methanol in the mobile phase was
changed (1-91%) at various rates from 0.75 to 6.0%/min, and the Kow value for two
test glucuronides (phenyl- and naphthyl-) were plotted vs the percent methanol in the
mobile phase at the time of analyte elution (Figure 2.). The Kow value for phenyl
glucuronide was found to be insensitive to the change in percent methanol, eluting
with 49.5% methanol in a 6%/min gradient and 6.7% methanol in a 0.75%/min
gradient, but having a Log Kow value of -0.69 in each case. Naphthyl glucuronide,
however, was found to be sensitive to the change in percent methanol, eluting in
76.2% methanol in a 6%/min gradient with a Log Kow value of 1.10 which increased
Because Log
to 1.39 when it eluted in 21.9% methanol from a 0.75%/min gradient.
Kow values for both compounds changed little when the gradient was slowed from
1.0 to 0.75%/min, and because laboratory time could be saved using the faster
gradient, the 1.0%/min gradient was selected to be used for the determination of Kow
for the other glucuronides. Values reported are as measured, rather than extrapolated
to Kw. Repeated analysis of naphthyl glucuronide showed a variability of ±0.01 log
units, with a percent relative standard deviation of 0.16% (n=7). Results for the
remaining glucuronides are presented in Table 1 (mean values, n=3). The measured
values reported for p-aminophenyl- and p-hydroxyphenyl glucuronides are presented
as estimated less than values. This is because they eluted between the column void
volume marker, phenyl phosphate, and the first eluting standard, uracil. We were not
able to obtain a Kow value for phenyl phosphate from the literature, nor was CLOGP
able to calculate a value for this compound. Having a Kow value for phenyl
phosphate may have allowed us to better estimate the Kow value of these two
glucuronides. Log Kow values for the other glucuronides ranged from -0.69 for
phenyl glucuronide to +1.79 for p-phenylphenyl glucuronide.
Glucuronide Kow values were evaluated in two ways, by comparison to their Kow
values calculated by CLOGP, and by correlation to Kow values of a series of
analogously substituted aromatic hydrocarbons (Table 1.). Measured and calculated
Kow values were highly correlated (r2=0.9402); however, calculated values were
consistently lower, averaging 0.86 units lower. Similarly, a good correlation was
found between the glucuronide Kow values and those for the aromatic hydrocarbons
(r2=0.9288).
Phenyl- and naphthyl glucuronide were also chosen for an assessment of ion-pair
formation upon Kow. In an organic solvent-water system, an organic acid may
partition into the organic phase as either a free anion, or as an neutral ion-pair, which
will affect the apparent Kow of the acid (Strathmann and Jafvert 1998). The Kow
values for the two test compounds were determined again using a methanol gradient
of 1%/min, however, the buffer (sodium phosphate) concentration was first lowered
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*
HPLC gradient 100%A to 100%B at 1.0%/min, A=1% methanol, B=91% methanol,
pH 6.8-7.0, 1.0mM sodium phosphate.**
1979.
Leo and Weinninger 1988.*** Hansch and Leo
Table 1. HPLC determined and CLOGP calculated Log Kow values for a series of aryl
glucuronides, along with Log Kow values for analogous substituted PAHs.
Figure 1. Chromatogram of HPLC column calibration compounds with phenyl- and
naphthyl glucuronide (lower trace) and p-chlorophenyl glucuronide (upper trace).
Chromatographic conditions were 1-91% methanol at 1 %/mm, 1 mM sodium phosphate,
pH 6.8. UV spectrum (left insert) and ESI/MS (right insert).
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to 0.5mM and then raised to 25mM for each determination. The three Kow values
for phenyl glucuronide were - 1.20, -0.69, and -0.11, while for naphthyl glucuronide
the values were 1.12, 1.40, and 1.61, for 0.5, 1.0 and 2.5mM, respectively (Figure 3.).
This experiment showed that Kow for the anionic glucuronides was very sensitive
to ion-pair formation with cationic sodium. The concentration of sodium in blood
of rainbow trout has been determined to be about 145mM (McDonald and Milligan
1992). Kow values for either phenyl or naphthyl glucuronide can not be determined
at such a high concentration of sodium because sodium phosphate will begin to come
out of solution at concentrations above about 5mM in mobile phase B (91%
methanol). By using a stronger organic modifier, such as acetonitrile, it is possible
to decrease the percent of organic modifier in mobile phase B while keeping the
retention time for analytes approximately the same. The concentration of the buffer
salt in mobile phase B may then be increased, thus allowing the determination of
Kow values to be done at higher concentrations of sodium. It was necessary,
however, to first assess the effect of acetonitrile on Kow. Using mobile phases A:1%
acetonitrile, and B:51% acetonitrile, containing 1mM sodium phosphate, Kow values
for phenyl and naphthyl glucuronide were assessed at different percentages of
acetonitrile in the mobile phase at the time of elution of the analyte in an experiment
similar to that conducted with methanol (Figure 2.). Log Kow values for phenyl
glucuronide eluting between 4.3 and 26.2% acetonitrile increased just slightly from
-0.66 to 0.78, and were similar to the values obtained using methanol. Log Kow
values for naphthyl glucuronide were lower than those obtained using methanol,
ranging from 0.68 at 40.8% acetonitrile to 1.16 at 13.0% acetonitrile. As with the
methanol system, Kow values did not change much when the gradient was slowed
from 1.0 to 0.75%/min. To be comparable to the methanol data, a 1%/min gradient
was then chosen to continue the ion-pair formation study. The concentrations of
sodium used were 0.5, 1.0, 2.5, 5.0, and 20.0mM. Results are shown in Figure 3.
For both of the test glucuronides the change of Log Kow between 0.5 and 5.0mM
was large, going from -1.08 to -0.04 for phenyl glucuronide, and from 0.91 to 1.49
for naphthyl glucuronide, but then became asymptotic at 0.35 and 1.61, respectively,
by 20mM.
In summary, Kow values presented in Table 1 were generated using instrumental
conditions that should be easily reproducible in other laboratories so that partitioning
data for other glucuronides may be compared to those presented here. The measured
values seem reasonable because they correlate well with mathematically calculated
values; are approximately 2.0-2.5 log units lower than the values for precursor
aromatic hydrocarbons; and, correlate with Kow values for structurally similar
aromatic hydrocarbons. Kow values for the glucuronides were found to be very
sensitive to ion-pair formation, which may become very important if partitioning
needs to be determined in a matrix such as blood where variations the concentration
of cationic species such as Na+ and Ca++ may become important in the complexation
with anionic organic species.
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% Organic Modifier
Figure 2. Log Kow values for phenyl- (pg) and naphthyl glucuronide (ng) vs percent of organic modifier
Data generated from a linear
in the HPLC mobile phase at the time of elution of the glucuronide.
gradient (100%A: 1% methanol (MeOH) or 1% acetonitrile (ACN) to 100%B: 91% MeOH or 51% ACN,
respectively, in 15, 30. 60, 90, or 120 min.
Figure 3. Log Kow values for phenyl- (pg) and naphthyl glucuronide (ng) eluted with either methanol or
The percent organic modifier
acetonitrile vs sodium phosphate concentration in the HPLC mobile phase.
change in the mobile phase was 1 %/min Methanol changed from 1-91%. and acetonitrile changed from
1-51%.
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