Download Urine Color Test for the Detection of

Urine Color Test for the Detection
Phenothiazine
of
Compounds
Irene S. Forrest and Fred M. Forrest
N PREVIOUSLY
REPORTED
urinary
color tests for chlorproinazine,
promazine, and mepazine
(1, 2) a test solution
containing
ferric chloride
in dilute
sulfuric
acid was mixed with the urine sample,
and the
color reaction
resulting
from the respective
drug metabolite
and the
test solution
was immediately
read against
prepared
color charts.
These reactions
were specific for the indicated
drugs which, in psychiatric
hospital
practice,
are administered
in daily doses usually
ranging
from 100 to several
thousand
milligrams.
In testing for the more recently
developed
drugs containing
either
a piperazinyl
group
such as prochlorperazine
(Compazine),
perphenazine
(Trilafon),
thiopropazate
(Dartal),
their
trifluoromethylated
derivatives
Stelazine
and Prolixin,
or trifluoropromazine
(Vesprin),
and for smaller
amounts
(less than 100 mg. per day) of
chlorpromazine
(Thorazine),
promazine
(Sparine),
or mepazine
(Pacatal),
a more sensitive
reagent
for this entire
group
of compounds was devised.
This new test solution
consists
of 5 parts of 5
per cent ferric chloride,
45 parts of 20 per cent perchioric
acid, and 50
parts of 50 per cent nitric acid (subsequently
referred
to as FPN).
This test solution
produces
immediate
color reactions
with the urines
of patients
who have ingested
any phenothiazine
compound,
including
the various
antihistaminic
compounds
of this group.
In this test, 1
ml. of FPN test solution
is mixed with 1 ml. urine.
In the lowest
phenothiazine
levels, corresponding
to drug intakes
of 5 to 20 mg.
per day, the yellow urine color turns to a light pinkish-orange,
while
From
tIme Research
Laboratory
and Acute
Service,
Brockton,
Mass.
Development
of this test was carried
out witim funds
Squibb
& Sons which is gratefully
acknowledged.
Veterans
Administration
froma a research
grant
Hospital,
from
E. R.
12
FORREST & FORREST
Clinical
Chemistry
daily drug doses of 25 to 70 mg. produce
reactions
in various
shades
of pink. Drug doses of 75 to 120 mug. daily yield increasingly
intense
shades
of violet, while doses of 125 rug, and more are reflected
by
deep purple
reactions
of increasing
opaqueness
and inklike appearance.
The color chart
(Fig. I) illustrates
the drug concentration
range and the color developed
upon addition
of the reagent.
The stability
of these color reactions
varies from a minimum
of
about 10 sec. for the lowest drug doses up to 30 mg., to 20 to 30 sec.
for the medium range of doses up to 73 mg., and up to several minutes
for the higher drug doses.
While FPN reagent
does not yield the optimum
scale of color development
for each of the individual
drugs tested,
it proved
useful
as a general
group reagent,
indicating
absence or presence
of phenothiazine
compounds
and allowing
a rough
estimate
of quantity
of
drug ingested.
In patients
on continuous
administration
of phenothiazine
drugs,
any urine specimen
is suitable
for the test. In the case of extremely
small daily doses of below 20 mg., or in the case of administration
of a single small dose, the period of between
2 and 5 hours after administration
is the optimum
testing
time.
SENSITIVITY OF THE FPN REACTION
From simultaneously
undertaken
studies
on the excretion
period
of phenothiazine
compounds*
in psychiatric
patients
after
discontmuation
of administration,
we found by means
of this and other
sensitive,
specific reactions
that urinary
excretion
extends
over a
period of more than 12 weeks rather than the 4 to 7 days indicated
in
the literature
(3, 4). After a single dose of 10 mg. of a phenothiazine
drug, this test on 1 ml. of urine, carried
out between
2 and 5 hours
after
drug administration,
shows a distinct
pink reaction.
From
parallel
quantitative
determinations
according
to a modification
of
the method indicated
by Salzman
and Brodie
(5) we know that 1 ml.
of urine in this instance
contains
less than 1 g. of sulfoxide,
the
major metabolite,
which does not yield a color reaction.
Therefore,
there seems to be present,
in addition
to the sulfoxide,
an appreciable
amount
of other metabolites,t
responsible
for the color reaction.
Thus a fraction
of a microgram
of intermediary
drug metabolite
is
demonstrable
by FPN.
*
Study
now
in progress
tA quantitative
of their metabolites
in this
method
for
is currently
hospital.
the determination
being developed.
of
urinary
phenotimiazine
drugs
and
all
Vol. 6, No. I, 1960
DETECTION
+
++
5-20 mg.
20-70 mg.
Fig. 1. Color
chart
the test, add 1 nil. FPN
mixture
in the test tube
test.
A
Shades
higher
chloride
OF PHENOTHIAZINE
for urine
test
+++
for
70-120 mg.
small
amounts
13
COMPOUNDS
of phenothiazimme
++++
120 mg. & over
drugs.
To performn
test solution
to 1 ml. urine, mix gently,
and match the color of the
against
tIme clmart. Read within 20 sec. No color indicates
a negative
slight darkening
without
formation
of pink or violet shades is considered
negative.
of violet
darker
than indicated
for the --f-}--jlevel represent
correspondingly
concentrations
of drug.
The FPN
test solution
is composed
of 5 ml. 5% ferric
solution,
45 ml. 20% perchloric
acid, and 50 ml. 50% nitric acid.
14
FORREST & FORREST
RELIABILITY OF TEST AND POTENTIALLY
Clinical
Chemistry
INTERFERING FACTORS
In more than 1500 urine specimens
tested,
we have seen no false
negatives.
In extremely
dilute specimens
(after fluid intake of more
than 3 L. per day) and a testing time of more than 5 hours after drug
administration,
a false negative
might
result,
however.
All other
apparently
false negative
tests could be traced to the patients’
failure
to ingest the medication.
(With regard
to the problem
of psychiatric
patients
“cheeking”
the drugs, see Refs. 1, 2, 6, 7, 8, and 9.)
We have seen between
1 and 2 per cent false positives
in approximately
500 control
urine tests of patients
and personnel
in the absence of phenothiazine
drugs.
All but one could be traced to elevated
urinary
levels of bile metabolites,
particularly
urobilinogen,
which
yields similar
color reactions
with FPN.
Thus in patients
with impaired liver function,
this test can not be uncritically
applied.
Nonphenothiazine-derived
drugs-e.g.,
reserpine,
barbiturates,
ineprobamate,
the various
energizers,
and vitamins-did
not show
any color development
with the reagent,
nor did they interfere
with
the test.
Color reactions
similar to those obtainable
with FPN are obtained
with 50 per cent nitric acid as the test solution.
However,
the resulting color tests are very unstable
and actually
fleeting
at low drug
levels, a fact which complicates
their evaluation.
Similar
difficulties
were encountered
in the method
of Neve (7), who uses a mixture
of
nitric acid and sodium nitrite
solution
as test reagent.
While FPN is a sensitive
group reagent
for all phenothiazine
compounds that permits
a rough conclusion
on the total quantity
of such
drugs present
in urine, no conclusion
with regard
to the specific drug
or mixture
of drugs can be drawn.
Some of the more recently
introduced drugs derived
from phenothiazine
show optimum
color development
suitable
for semiquantitative
readings
against
color charts
with different
reagents.
Rapid
urinary
tests for these individual
drugs and their optimum
reagents
(various
heavy-metal
salts in concentrated
hydrochloric
acid), with pertinent
color charts,
have been
reported
by us (8, 9).
In view of the above-mentioned
protracted
urinary
excretion
time
after discontinuation
of long-term
drug administration,
it should be
considered
that any urinary
drug level may result
from a recently
ingested
small dose, or from a high dose possibly
preceding
the test
by several
weeks.
Hence it is not possible
to determine
by a single
(e.g., pink) FPN reaction
whether
a patient
took, for instance,
20
Vol. 6, No. I, 1960
DETECTION
OF PHENOTHIAZINE
mg. of a drug on the day of the test,
800 mg. 2 weeks before.
15
COMPOUNDS
75 mg. on the previous
day,
REFERENCES
1.
2.
Forrest,
Forrest,
F. M., and Forrest,
I. S., Am. J. Psychiat.
113, 931 (1957).
F. M., Forrest,
I. S., and Mason, A. S., Am. J. Psychiat.
114,
3.
4.
5.
6.
7.
8.
9.
Citterio,
Fedorov,
Salzman,
Pollack,
Neve, H.
Forrest,
Forrest,
C., Lay.
N. A.,
N. P.,
B., Am.
K., J.
F. M.,
F. M.,
931
Neuropsychiat.
20/2,
201 (1957).
and Shnol, S. E., Zh. Nevropat.
Psikhiat.
56, 139 (1956).
amid Brodie,
B. B., J. Pharin.
4. Exp. Ther. 118, 56 (1956).
J. Psychiat.
115, 77 (1958).
Ment. Sc. 104, 488 (1958).
Forrest,
I. 5., and Mason,
A. S., Ani. J. Psychiat.
115, 1114
Forrest,
I. S., and Mason,
A. S., Am. J. Psychiat.
116, 549
(1958).
(1959).
(1959).
or