until
the
pink
color
disappears.
An
additional
560 ml of concentrated
H4S04 is added,
and the solution
is stirred
overnight
to cool. This
A Stable Stock Arsenic Pentoxide
Reagent for Use in Preparing
Diacetyl Monoxime Reagent for a
Urea Nitrogen Procedure
solution
is diluted
water.
The solution
To prepare
months.
etyl
The
single-reagent
manual
method
for directly
determining
urea nitrogen
in serum
that was described
by our
laboratory (1) has been highly satisfactory.
One modification
in the preparation of the reagents
is a time saver,
and
will definitely
be of value
to
other laboratories
preparing
large volumes
of the diacetyl
monoxime
reagent.
In the method
as originally
described
(1) the diacetyl
monoxime
(2,3-butanedione
monoxime)
reagent
was prepared
by dissolving
10 g of arsenic pentoxide
(As2O5, Baker “Analyzed,” No. 0052; J. T. Baker Chemical Co., Phillipsburg,
N. J. 08865)
and 7.0 g of NaOH in about 800 ml of
distilled
water in a 1-liter Erlenmeyer
flask. This was heated
and stirred
to
dissolve
the arsenic
pentoxide.
Two
drops
of phenolphthalein
indicator
(10 g per liter
of methanol:water,
60:40
by volume)
was added
and
then
concentrated
H2S04
was added
until the pink color disappeared.
An
additional
28 ml of concentrated
H2SO4 was then added. This solution
had to be cooled to room temperature
and then 3.0 g of phenazone
me;
Mo.
Sigma
63178)
noxime
(antipyr-
Chemical
Co., St. Louis,
and 5.0 g of diacetyl
mo-
(Baker
Analyzed
Reagent)
were
added
and
the solution
was
stirred
until
completely
dissolved.
This was then diluted
to 1-liter with
distilled
water. This mixture
was stable for two months
when stored
at
4#{176}C
in a brown bottle.
For laboratories
involved
in large-
volume testing of serum
gen, the above reagent
urea nitrohas to be
made in much larger volumes
or on a
much
more frequent
basis.
Because
so much heat is evolved after the sulfuric acid is added,
we usually let the
mixture
cool overnight
before the reagent is completed.
A “stock
arsenic
pentoxide
reagent,”
which
can be prepared
in
bulk and stored at room temperature
until needed
in the diacetyl
monoxime reagent,
may be prepared
as follows: 200 g of arsenic
pentoxide
and
140 g of NaOH are dissolved
in 16 liters of distilled
H2O. This is heated
at about 80-90#{176}C
and stirred until solution is complete.
About 20 drops of
phenolphthalein
indicator
are added
and concentrated
H2S04
then added
with
may be stored
a polypropylene
container
temperature
for
as
long
the
monoxime
reagent,
arsenic
pentoxide
“stock
To the Editor:
to 20 liters
at
in
room
as
six
diac-
working
800 ml of the
reagent”
is
placed
in a 1-liter flask. Antipyrine
(3.0 g) and diacetyl monoxime (5.0 g)
are added.
The solution
is stirred
until
the components
dissolve,
then
diluted
to 1-liter with the “stock arsenic
pentoxide
reagent.”
This
reagent is stable for two months
when
stored at 4#{176}C
in a brown bottle.
The preparation
of the final reagent
takes about 20 mm, whereas if the reagent
is
prepared
without
the presence
ethchlorvynol
the extract
of
or serum.
If
solution
with
CHC13 is used for quantitating
the
concentration
of ethchlorvynol,
we
found that recoveries of ethchlorvynol
averaged
about
80% when based on
standards
taken
through
the
entire
procedure.
References
J. W., Hopkins,
N. E., Queen,
of phenazopyridine hydrochloride
(“Pyridium”)
on
determination
of ethchlorvynol
(“Placidyl”).
Clin. Chem. 18, 591 (1972). Letter
to the Editor.
2. Frings, C. S., and Cohen, P. S., Rapid
1. Cantrell,
C. A., and
C. S., Effect
Frings,
colorimetric
determination
method
for the
of ethchlorvynol
in serum and urine.Amer.
54, 833 (1970).
quantitative
(Placidyl)
J. Clin. Pathol.
Christopher
S. Frings
Cecelia A. Queen
Medical
Laboratory
Associates
1025 S. 18th St.
Birmingham,
Ala. 35205
Use of “Low-Lead” Vacutainers
for Blood Collection
Reference
or absence
in urine
of the final
utilizing
the
“stock
arsenic
pentoxide
reagent,”
the total time will be about 5
h. We recommend
this modified
reagent preparation
procedure
for those
laboratories
utilizing
larger
than
1liter quantities
of the reagent.
1. Foster,
detecting
in ALA-D
Assay
L. B., and Hochholzer,
J. M., A
directly To the Editor:
determining
urea nitrogen in serum. Clin.
In the assay of blood for lead it is esChem. 17, 921 (1971).
sential
that contamination
be miniTed W. Fendley
mized, and certified
“minimum”
lead
content
tubes
(Becton-Dickinson,
Manuel Price
single-reagent
manual
method
Christopher
for
S. Frings
Medical Laboratory
Associates
1025 S. 18th St.
Birmingham,
Ala. 35205
Elimination of Interference by
Phenazopyridine
Hydrochloride
(“Pyridium”) in Determining
Ethchlorvynol (“Placidyl”)
To the Editor:
Our laboratory
(1) recently
reported
that
phenazopyridine
hydrochloride,
a drug widely used for the symptomatic relief of irritation
of the lower
urinary
tract
mucosa,
causes
falsely
positive
results
with the colorimetric
method
of Frings and Cohen
(2) for
the determination
of ethchlorvynol
(“Placidyl,”
Abbott).
We have found that certain
organic
solvents
will remove
the interference
caused
by phenazopyridine
hydrochloride.
If the solution
in the final
step of the ethchlorvynol
procedure
(2)
containing
the
“ethchlorvynol
chromogen”
is shaken
with an equal
volume
of CHC13, the pink chromogen caused
by phenazopyridine
hydrochloride
stays
in the
aqueous
phase and the pink chromogen
caused
by ethchlorvynol
is extracted
into the
CHC13 layer.
This information
should
be useful
for
those
laboratories
using
this
method
as a qualitative
“screen”
for
CLINICAL
Cat.
No.
L3200
x F313)
are
often
used. An alternative
test for lead exposure is the measurement
of the activity
of the erythrocyte
enzyme
#{244}amino
laevulinate
dehydratase
(EC
4.2.1.24)
(ALA-D)
(1). Activity
is reliably depressed
in inverse proportion
to the blood lead concentration.
However, it has been our observation
that
bloods collected
in the above tubes do
not show the expected
decrease
in
ALA-D
activity
in response
to increased
concentration
of lead in blood. Bloods
collected
or stored
in these certified
“low-lead”
tubes
show
consistently
higher ALA-D activities
than do bloods
collected
in green-stoppered
“Vacutamer”
tubes
or heparinized
plastic
ones.
The
15 blood
ALA-D
samples
activities
were
for
which
determined
(Table
1) were from those submitted
to our laboratory
for analysis.
A portion
of the submitted
sample
was
transferred
to
a
brown-stoppered,
“low-lead”
tube for approximately
hour. All samples
were kept iced
parallel
determinations
were run
an
and
by
(2).
These split samples
contained
an increased
amount
of heparin,
but studies showed
that
increased
heparin
does not produce
the enzyme
stimulation. The percentage
increase
in activity
is much
greater
in those samples that have abnormally
high lead
concentrations
and depressed
enzyme
the
method
CHEMISTRY,
of
Burch
and
Siegel
Vol. 19, No.9,1973
1087