CLIN.CHEM. 28/7,1457-1460(1982)
Simple, ReliableChromatographicMeasurementof Oxalate in Urine
Antonio Di Corcia,’ Roberto Samperi,1 Giuliana Vinci,1 and Giuseppe D’Ascenzo2
In thisassay foroxalateinurine,oxalateisadsorbed from
the urineonto graphitizedcarbon black (Carbopack B).
Afterdesorptionand removal of the solvent,oxalicacid
is gas-chromatographically
measured after being deriva-
tizedwithBF3/methanol.Chromatography is on Carbopack
B/polyethylene glycol (M. 20 000),93.7/6.3by weight.The
lower limit of detection of urinary oxalate is about 3 mg/L
(CV
3.6%). A seriesofoxalatedeterminationsin24-h
urinesamples of 15 subjectsgave a mean of 47.1 (SD
15.4) mg/24 h, with an analytical recovery of 97.4% (SD
3.0%, range 93.6-102.4%).
Total analysis time for one
sample is about 2 h.
AddItIonal Keyphrases:
adsorption
vatization
gas chromatography
desorption
den-
#{149}
Determination
of urinary oxalate is of much interest for the
diagnosis of various types of primary hyperoxaluria.
Knowledge of the oxalate content of urine from patients with calcium
oxalate nephrolithiasis,
correlated with other data, can also
aid urologists in clarifying which biochemical and (or) physiological mechanisms are operative in stone formation.
Although there has been considerable work on methods for
urinary oxalate measurement,
almost none of these combine
simplicity, rapidity, and reliability.
Enzymic methods
(1,2) are highly sensitive and require no
sample pretreatment
but are time consuming and expensive
(3). Colorimetric determinations
(4) are simple to perform but
are affected by several positively interfering substances that
can cause overestimation
of urinary oxalate (5).
Gas chromatography
(GC), being both specific and sensitive, has been extensively exploited for determination
of oxalate in both urine and serum (5-7). Nevertheless,
many of
the GC methods reported involve time-consuming
samplepurification procedures, such as precipitation.
Adsorbing materials for cleanup of biological samples are
increasingly attracting attention for determination
of compounds of biomedical interest (8-10). Column techniques
for
this are rapid, reliable, and simple, and offer additional selectivity when combined with GC analysis.
Here we describe an accurate, rapid, and simple method for
determining oxalate in urine. Samples are passed through a
colunm filled with graphitized carbon black as adsorbent. The
retained oxalate is eluted with acidified methanol. This solvent is then evaporated and the residue, derivatized with BF3
in methanol, is measured by GC.
Materials and Methods
We obtained
24-h urine specimens from 15
with 30 mL of toluene in the container as preserva-
Materials.
subjects,
Istituto di Chimica Analitica, Universit#{224}
Degli Studi di Roma,
00185 Roma, Italy.
2 Istituto
Chimico, Universit#{224}
di Camerino, Italy.
Received Feb. 8, 1982; accepted April 6, 1982.
tive. Oxalic acid (99.5%), 2-heptanone (the internal standard),
and boron trifluoride (100 mL/L of methanol) derivatizing
agent were all obtained from Fluka AG, Switzerland. Graphitized carbon black (Carbopack B, 80-120 mesh) was from
Supelco Inc., Bellefonte, PA 16823. Polyethylene glycol (Mr
20 000) was from Carlo Erba, Milan, Italy.
We used the Carlo Erba Model GI gas chromatograph
equipped with a flame ionization detector, and we quantified
peaks with a Shimadzu Model Chromatopac
C-E 1 B integrator. The gas-chromatographic
column adopted for quantitation was glass, 2 m X 2 mm (i.d.), packed with the same
material used for the cleanup procedure-that
is, Carbopack
B-suitably
modified with added polyethylene glycol (PEG).
Preparation of the packing material and the column packing
procedure were as previously described in detail (11, 12); final
proportions
of materials are Carbopack/PEG
93.7/6.3 by
weight. The GC column was conditioned
overnight
at 250 #{176}C
with nitrogen. For the quantitation procedure, the column was
operated at 145 #{176}C
with nitrogen as carrier gas and with a dead
time of 25 s. The injection-port
temperature
was 180 #{176}C.
A
5-sL Hamilton syringe, Model 801, was used to inject the final
sample.
Procedure. Prepare the purification
0.6 g of Carbopack
B particles
column by suspending
in 0.1 mol/L aqueous HC1 and
into a 14 X 0.6 cm glass column
introducing the suspension
fitted with a Teflon stopcock. Pack the adsorbent by tapping
the column while passing distilled water through it, until the
column of carbon is 6.5cm high and the effluent water is at pH
6 (pH paper). Pass through the column, at a flow-rate of about
0.6 mL/min, 2 mL of the 24-h urine, previously diluted with
an equal volume of water and then acidified with concentrated
HC1 to pH 3 (pH meter). When the meniscus of the sample
reaches the top of the carbon, wash the column by passing
through 1.5 mL of water acidified with HC1 to pH 3. Use
methanol acidified with HC1 (0.1 mol/L) to desorb oxalic acid
from Carbopack B. Discard the first 4.6 mL of the effluent
from the column and collect the following 1.3 mL, which
contains oxalic acid, in a 10-mL air-tight screw-capped glass
vial fitted with a silicone-rubber
septum. The discarded
fraction consists of 1.2 mL of dead volume, 2 mL of diluted
urine,
urine
and 1.4 mL of the acidified
water
volume.
Evaporate
the collected
just following
fraction-mainly
the
methanol, with some water-under
a stream of nitrogen while
the tube is in a water bath maintained at 50 ± 1 #{176}C.
Derivatize
the residue by adding 0.1 mL of the methanolic BF3, capping
the vial, and incubating for 5 mm in a water bath at about 70
#{176}C.
After cooling the tube, add 10 L of 2-heptanone
solution
(1.11 gIL), then inject 0.6 tL onto the GC column.
Figure 1A shows a typical GC profile for an unsupplemented urine.
Calculations.
To calculate the amount of oxalate in urine
from the GC peak, prepare an oxalic acid/water/methanolic
HC1 standard solution and evaporate a known volume of it.
After derivatization,
add 10 sL of the 2-heptanone
standard
solution, make replicate injections, and measure the peak area
ratios of the dimethyl oxalate and the internal standard.
We
CLINICAL CHEMISTRY,
Vol. 28, No. 7, 1982
1457
A
B
E
x
I-
II-
4
a
#{243}
468
TIME(min)
Fig. 1. Gas chromatograms of a purified and derivatized urine: A, diluted with an equalvolume of water;B, diluted 20-fold with
aqueous HCI (pH 3) to evaluate the minimum detectable amount of oxalate
IS.,
Internal std
repeated this procedure four times, varying the volume of the
oxalic acid standard solution. Calculate the response factor
(R.F.) for oxalic acid vs the fixed amount of internal standard
used by dividing the amount of oxalic acid by the relative peak
area obtained. When the concentration
of the internal standard solution is 1.11 g/L, the R.F. value is 0.0659.
Then calculate the amount of oxalate in 1 mL of urine (the
urine volume effectively submitted to analysis) by the expression:
glLofurine
method,
we prepared
a 24-h urine
sample
as described
in the
previous section, and divided it into eight aliquots.
Three of these aliquots were supplemented
with known,
increasing amounts of oxalic acid. Four aliquots were further
diluted
with various
known
amounts
of aqueous
HC1, pH 3.
One aliquot was left unmodified.
Each aliquot was analyzed
six times, and the results are shown in Table 1. The coefficients of variation ranged from 1.5% for medium-concentration urines to 3.6% for high-dilution
urine. The medium CV
was 2.5% over the range of oxalate concentrations
considered.
The limit of sensitivity
at which
oxalate
could
be accurately
measured
where T is the mean recovery; A and Ais are the respective
peak areas for oxalate and the internal standard; 88 is the
relative molecular mass of the oxalate anion; and 90 is the
relative molecular mass of oxalic acid. This latter correction
is introduced to account for the fact that oxalic acid is used
to calculate the R.F. value.
Analytical
Results and Discussion
Precision
and Limit of Sensitivity
To evaluate the precision and the limit of sensitivity
1458 CLINICALCHEMISTRY,Vol.28,No.7, 1982
(CV 3.6%) was about 3 mg/L. At this concentration,
a well-defined
chromatographic
peak for oxalate could be still
obtained (Figure 1B). The percentage recovery was found to
be independent
of the amount of oxalate in the urine. Evidently the adsorbent used for the cleanup procedure was not
saturated.
of the
Recovery
and Normal Values
Recovery
was evaluated
by adding known amounts of oxalic
acid to 24-h urine specimens from 15 different subjects and
analyzing
them. Recovery was 97.4% (SD = 3.0%, range
Table 1. Results of Analyses (n 6) of a Urine
Supplemented with Oxalic Acid or Diluted with
Aqueous HCI (pH 3)
=
Table 2. Analytical Recovery of Oxalate under
Various Analytical Conditions
OxaIIc acId In urhie, a mg/L
Found
Mean (SD)
CV, %
0
42
84
64.7 (1.8)
103.7 (2.3)
146.3 (2.2)
2.8
2.2
1.5
97.2
98.4
300
357.4(5.7)
1.6
98.0
Added
0, dil.
2foldb
0, dll.
5-fold
0, dll.10-fold
0, dil. 20-fold
31.3(0.6)
12.8(0.45)
6.5(0.15)
1.9
3.5
2.3
3.3 (0.12)
3.6
‘Diluted wIth an equal volume
wIth aqueous HCI, pH 3.
Recovery,
of water before analysIs (see text).
93.6-102.4%). The daily mean oxalate excretion
(SD 15.4 mg, range 31.7-91.8 mg).
5DIIuted
was 47.1 mg
As reported elsewhere (13, 14), the selectivity of liquidmodified GC can be changed by varying the proportions
of the
liquid on the adsorbent surface. We found 6.3% PEG/modified
Carbopack B to be the most selective packing material for GC
quantitation
of oxalic acid. Definitive evidence of this was
achieved by connecting the chromatographic
column to a mass
spectrometer.
For 15 different urine samples, the fragmentation patterns of the peaks supposedly representing oxalate
in urine were identical to the pattern for authentic dimethyloxalate, no additional peak in the mass spectrum being observed.
Internal standard was not added at the beginning of the
extraction and derivatized, because no synthetic oxalate-like
compound
was eluted together with oxalic acid from the purification column. Of many compounds tested, 2-heptanone
appeared to be the most suitable internal standard, because
its retention time is near to that of dimethyl oxalate and its
peak did not overlap with peaks for endogenous compounds
in urine. Usually, to obviate incomplete conversion of oxalic
acid to its corresponding ester, an acidic compound is used as
internal standard by adding it before the derivatization
procedure. This expedient was found to be unnecessary because
we observed that, under our experimental
conditions,
residual
amounts of oxalic acid were converted to dimethyl oxalate in
the injection port of the GC apparatus.
Storage
The effect of storage on the oxalate concentration
in urine
was evaluated. A 24-h urine, collected with toluene as preservative and stored at room temperature, was analyzed seven
times during three months. We saw no significant change in
the oxalate concentration.
The day-to-day CV was 5.3%.
AnalyticalOptimization Studies
We made a set of measurements
on a 24-h urine sample
from a selected subject, with analytical conditions described
in the procedure section each time being modified by changing
only one sequential variable. This was done to establish optimum analytical conditions for accurate, rapid oxalate determination
and to ascertain which conditions were critical.
2 summarizes
the results.
As can be seen, there was considerable
As reportedintext
Undiluted urine
Sample atpH 2.5
%
Specificity
Table
Recovery, %
(n = 3)
ConditIons
loss of oxalate if the
93.6
53.0
62.3
75.5
83.2
79.3
81.2
93.6
Sample at pH 3.5
Without water washes
Bath temperature at 60 #{176}C
50 zL ofBF3/CH3OH
Time ofderivatization:
5 mm
dilution step for this particular urine was omitted, but for
other urine samples examined, oxalate recovery was not
markedly increased after sample dilution with water. The
reason for this behavior is not clear to us. Perhaps this urine
contained
substances
capable of capturing oxalate. If so, this
ability was strongly dependent upon concentration,
because
a simple one-to-one dilution with water sufficed to overcome
such activity. Accordingly, we dilute urines before analysis,
to eliminate the possibility of occasionally obtaining erratic,
low values for oxalate concentration.
The losses observed when the pH of urine was adjusted to
values other than pH 3 may be accounted for by two considerations.
First,
in an acidic
medium
some surface
sites of GC
are rearranged to form salts that can exchange anions (15).
This rearrangement
occurs when Carbopack B particles are
suspended in acidified water before the purification column
is prepared. Second, at pH 3, oxalate is almost exclusively
present as the HC2O anion, while the H2C2O4 or C20r forms
are partly present at pH values of 2.5 and 3.5, respectively.
From these considerations
and on the basis of the experimental observation, it follows that oxalate is retained by the
Carbopack
surface by an anion-exchange
mechanism. Moreover, oxalic acid is not adsorbed by the adsorbent and the
C20r anion is so strongly bound on the carbon surface that
the HC1/methanol solvent mixture cannot remove it.
Low analytical recovery on omitting the water-washing
step
before addition of acidified methanol may be ascribed to the
presence in urine of substances that water cannot remove from
the column but that, under our experimental conditions, are
eluted with methanol, interfering during the derivatization
of oxalic acid.
To optimize analytical conditions in terms of rapidity and
sensitivity, measurements
were made by varying the bath
temperature
for solvent removal, the duration of derivatization, and the volume of BF3/CH3OH needed to prepare dimethyl oxalate. Bath temperatures exceeding 50#{176}C
caused loss
of sample, probably owing to some decomposition
of oxalic
acid. Derivatizations
longer than 5 mm were unnecessary.
Volumes of BF3/CH3OH lower than 0.1 mL caused incomplete
derivatization
of oxalic acid, probably owing to the presence
of traces of water in the residue, which partly decompose
BF3.
We thank
F. Bruner
(Urbino,
Italy)
for GC-MS measurements
and
Miss Doralba Berardo for assistance with the manuscript.
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CLINICAL CHEMISTRY,
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