A Simple Method for the Quantitative
Determination
of N-Acetyl-p-Aminophenol
(APAP) in Urine
Richard M. Welch and A. H. Conney
A simple method is described for the rapid quantitative analysis of N-acetyl-p-aminophenol (APAP) in urine. APAP and its conjugates present in the urine following the
ingestion of acetophenetidin, APAP or acetanilid, are hydrolyzed with acid to p.
aminophenol. This compound is coupled with phenol in the presence of hypobromite to
form an indophenol dye whose concentration is determined spectrophotometrically.
Application of this method to a study of 3 human subjects treated with acetophenetidin is described.
drugs,
such as acetophenetnhn
and
(APAP),
which give rise to conjugated
APAP
in the urine, has stimulated
an interest
for a simple and rapid method
which could be routinely
used in clinical
laboratories
for the detection
and quantitation
of these drugs and their metabolites.
The need for
such a test has been pointed
out by recent
reports
indicating
that
the abuse
of certain
of ti1ese analgesic
drugs
may Iea(l to Ihephropathies
(1,2).
The method previously
described
by Brodie and Axelrod (3,4)
for the determination
of conjugated
APAP
involves
acid
hydrolysis
followed
by extraction
of tile resulting
p-aminophenol
into
ethyl ether and then its extraction
from the organic
solvent into dilute
acid. The p-aminophenol
was then reacted
with phenol in the presence
of sodium hypobromite
to form an indophenol
dye whose concentration
was determined
in a spectrophotometer.
The method presented
here for the determination
of APAP
in urine
is simple enough for routine
use in the clinical laboratory.
The extraction of APAP
or p-aminophenol
into organic
solvent
described
by
Brodie
and Axelrod
has been eliminated.
APAP
and its conjugates
HE
\Vi1)ESPIIEAD
USE
of analgesic
N-acetyl-p-aminophenol
From tIme Wehlcome Research
Laboratories,
Burroughs
Welleonie
hoe, N. Y., and the Department
of Pharmacology,
Albert
Einstein
N.Y.
Received
for pubhicatiols
July 19, 1965;
accepted
for publication
I 064
& Co. (U.S.A.)
Inc.,
College of Medicine,
Oct.
13, 1965.
TuckaBronx,
Vol. II,
No. 12, 1965
N-ACETYL-p.AMINOPHENOL
1065
IN URINE
present
in urine are hydrolyzed
with acid to p-ammophenol
which is
coupled directly
with phenol ill the presence
of hypobromite
to form an
ilidopileilol
dye. A positive
test will be obtained
following
the ingestion
of certain
commonly
used analgesics
such as acetOl)henetidin,
APAP,
or acetanili(l
since these drugs
give rise to conjugated
A PAP in the
lirille.
Materials and Methods
Reagents
1.
4.ON HG1
to 100 ml.
lilte
Add 33.2 ml. of coiic. HC1 to distilled
2. O.20N NaOH
to 1000 ml.
,]. 1% w/v
phenol
Dissolve
8 gin, of NaOH
in distilled
water,
water,
and dilute
and
di-
J)issolve
1 ml. of ]iquefied phenol (Mallinckrodt
88% solution)
in distilled
water, and dilute to 88 ml.
4. 2N sodium
carbonate-bromine
solution
1)issolve
10.6 gm. of anllydrOus sodium carbonate
in distilled
water, and dilute to 100 ml. Add
15 ml. of a bromine-saturated
water solution
to 100 ml. of the sodium
carbonate
solution.
Bromine
solution
is
prel)ared
by allowing
excess
liquid bromine
to stand in distilled
water for at least 24 hr. The mixtiire is occasionally
shakeii and can be stored indefinitely.
Reagents
3 and 4 should be freshly
prepared
on the day they are to
A.R.;
be used.
5.
drug
Standard
solutions
solutions
containing
For the preparation
of
a standard
curve,
100, 200, 300, 400, 600, and 800 g./rnl.
are
p repa red.
Procedure
Add 1 ml. of urine to a test tube (150 mm. by 20 mm.) marked
at 10
Add 4 ml. of 4.ON HC1 followed by 1 ml. of distilled
water
or 1 ml.
of a standard
drug solution
to give a final volume of 6 ml. Stopper
the
test tube with a loosely fitting glass stopper
to prevent
evaporation
and
ml.
place in a boiling water bath for 1 hr. After tile boiling procedure,
add
distilled
water to give a final volume
of 10 ml. so that each milliliter
represents
0.1 ml. of urine.
Then pipet 1 ml. of the solution
into a 20-mi.
colorimeter
cuvet followed
by 10 ml. of a freshly
prepared
mixture
which contains
80 ml. of 0.2N NaOH, 10 ml. of 1% phenol, and 10 ml. of
the carbonate-bromine
reagent.
Mix gently by tapping
the tube, and
allow to stand
for 40 miii. for maximum
development
of blue color.
Frine containing
200 gig. of APAP per milliliter
gave an absorbance
of
0.215 at 620 m when 1 ml. of urine was analyzed
by the above method.
One milliliter
of drug-free
urine (analyzed
by the above method)
from
1066
WELCH
5 individuals
in a Beckman
gone through
& CONNEY
had absorbances
ranging
DU spectrophotometer*
the entire procedure.
Clinical
from
against
Ckemslry
0.025 to 0.045 when read
a water blank which had
Experimental
Various
milliliter
resulting
concentrations
of these urines
standard
curve
of APAP
were added to blank urine.
One
was analyzed
by the above method,
and the
is illustrated
in Fig. 1. For precise
quantita-
Fig.
::
a.
tions
/
200
0
0
200
Various
and
concentraabsorbance
at 620 mgi. One milliliter
of
blank urine or urine containing
various
concentrations
of added
APAP
was analyzed
by procedure described
in text.
40c
a.
1.
of APAP
.600
.800
ABSORBANCE AT 620 Mu..
.400
IOO
.20
tion of the APAP
present
in urine, standards
of APAP
should be run
with each experiment.
A 24-hr. urine sample
(1000 ml.) from an individual who had taken a tablet containing
a total of 325 mg. of APAP
was analyzed
for APAP
and its conjugates
according
to the method described
above.
A 1-ml. aliquot
when analyzed
by this
procedure
gave an absorbance
of 0.295 (10 times the value of drug-free
urine),
showing,
according
to Fig. 1, that 90% of the APAP
ingested
could be
accounted
for in the urine after acid hydrolysis.
in a similar
study of
an individual
who had taken 2 APC tablets
containing
a total of 325
mg. of acetophenetidin,
465 mg. of aspirin,
and 65 mg. of caffeine,
66%
of the acetophenetidin
could be accounted
for as APAP
in the urine
during
an 8-hr. period.
The method
was used in a further
study by measuring
the hourly
excretion
of APAP in the urine of 3 human subjects
after an oral dose
of 400 mg. of acetophenetidin.
The subjects
were given acetophenetidin by capsule 2 hr. before breakfast,
and urine collections
were made
every 2 hr. for the first 12 hr., followed
by one 12-hr. collection.
The
results
of this study
(Table
1) show that after an initial
2-hr. delay,
APAP is rapidly
excreted
in the urine and 70-80%
of the dose can be
*Beckman
Instruments,
Inc.,
Fullerton,
Calif.
Vol. I I, No. 12, 1965
Table
1.
CUMULATIVE
N-ACETYL-p-AMINOPHENOL
AMOUNTS
OF
ACETOPHENETIDIN*
PHENOL
PROM HUMAN
Acetophen
Hr.
after
dose
Subject
etidin
(nmale)
I
RECOVERED
recovered
as
Subject
(mote)
2
6
30
47
59
16
34
52
70
62
71
0.7
77
(400 mg.)
administered
orally
AS
N-ACEPYL-p-AMINO.
URINE
4
6
8
12
24
*Dose
1067
IN URINE
APAP
(%
of
dose)
Subje ct
3
(female)
5
31
55
70
77
79
by capsule.
accounted
for as APAP
within
24 hr. These results
agree quantitatively with an earlier
study
(4) which employed
a more time-consuming method.
Neither
urine
from
individuals
who had taken
600 mg. of aspirin,
nor blank urine containing
1 mg. of acetophenetidin,
1 mg. of 3-hydroxyacetophenetidin,
1 mg. of p-phenetidin
or 1 mg. of added
aminopyrine
per milliliter
of urine gave an absorbance
higher than was obtained
for
blank urine when analyzed
by the method
described
above.
The addition of 1 mg. of 2-hydroxyacetophenetidin
to 1 ml. of blank urine gave
an absorbance
slightly higher than was obtained
for blank urine.
However, the blue color characteristic
of indopheriol
dye formation
did not
appear.
References
1.
2.
3.
4.
Schreiner,
G. E., The nephrotoxicity
of analgesic
abuse. Ann. Internal
Mcd. 57, 1047 (1962).
Gilmnn,
A., Editorial:
Analgesic
nephrotoxicity.
Am. J. Med. 36, 167 (1964).
Brodie,
B. B. and Axelrod,
J., The estimation
of acetanilide
and its metabolic
products,
aniline,
N-acetyl
p-aminophenol
and p-aminophenol
(free
and total conjugated)
in biological fluids and tissues.
J. Pharinacol.
Exp. Therap.
94, 22 (1948).
Brodie,
B. B., and Axelrod,
J., The fate of acetophenetidin
(phenacetin)
in nman mmcl methods for the estimation
of acetophenetidin
and its metabolites
in biological
material.
I. Pharmacol.
Exp. T/ierap.
97, 58 (1949).