theophylline. (as aminophylline), caffeine, theobromine. (Sigma. Chemical. Co., St. Louis,. Mo. 63178), theophylline-7-acetic acid (Adams. Chemical. Co., Round.
Our assay
allows
centrations grams
accurate
down
4 mm;
determinations
to 1.0 mg/liter
maximal
sensitive enough pharmacokinetic
of serum
(peak
baseline
height
fluctuation
for clinical studies.
use,
1.5 mm).
and
high-performance (1974).
con-
in chromato-
appears
It is thus
suitable
for
liquid
J. Chromatogr.
chromatography.
99, 609
3. Blair, A. D., Forrey, A. W., Meijsen, B. T., and Cutler, R. B., Assay of flucytosine and furosemide by high-pressure liquid chromatogra-
phy. J. Pharm.
Sci.
64, 1334 (1975).
4. Nilsson-Ehle, I., Yoshikawa, T. T., Schotz, M. C., and Guze, L. B., Quantitation of antibiotics using high-pressure liquid chromatography: Tetracycline. Antimicrob. Agents Chemother. 9, 754 (1976). We thank the
Waters
AB, Gothenburg,
Associates
chromatograph
at our
Sweden,
for putting
5, Wold, J. S., Rapid liquid chromatography. (1977).
disposal.
analysis ofcefazolin in serum by high-pressure Antimicrob. Agents Chemother. 11, 105
6. Anhalt, J. P., Assay of gentaxnicin in serum by high-pressure liquid chromatography. Antimicrob. Agents Chemother. I 1, 651 (1977).
References
Nilsson-Ehle, I., Yoshikawa, T. T., Edwards, J. E., et al., Quantitation of amphotericin B with use of high-pressure liquid chromatography. J. Infect. Dis. 135, 414 (1977). 8. Hoehn, M. M., Murphy, H. W., Pugh, C. T., and Davis, N. E., Paper chromatographic techniques for the determination of cephalothin and desacetylcephalothin in body fluids. Appl. Microbiol. 20, 734 7.
1. Cooper,
M. J., Anders,
and high-speed acetylcephalothin 659 (1973). 2.
Buhs,
liquid chromatography in human serum and
R. P., Maxim, and their
cephalothin
M. W., and Mirkin, of urine.
extraction
cephalothin
Drug
and
Metab.
de-
Disp.
1,
T. E., Allen, N., et al., Analysis of cefoxitin, deacylated metabolites in human urine by
CLIN. CHEM. 24/2, 367-368
A. Owen
of Theophylline-7-acetic
assay
procedure
is described
for
individually measuring theophylline-7-acetic acid and theophylline in 2.0 ml of serum. Absorbance is a linear function of concentration over a range of 0.5-50.0 mg/liter theophylline-7-acetic
theophylline. were found phylline
acid
No endogenous that interfere with
determinations.
mg/liter
As with other spectrophotometric
Keyphrase:
AddItIonal
In practice, use because It is poorly
sociated
of its soluble
with
gastric
convulsions.
many
theophylline
is not a particularly
physical in water;
theophe-
easy drug
to
and pharmacological properties. furthermore, it is frequently as-
upset
and
palpitations,
In an attempt
derivatives
analogs, theophylline-7-acetic in 1949 (1 and is currently theophylline. This acidic perazine to form a neutral
and
to obviate
have acid,
)
administration.
for
drug assay
theophylline
causes
enteral
and 0.5-40
or exogenous compounds theophylline-7-acetic acid
assays, theobromine and, to a slight extent, interfere with theophylline determinations.
nobarbital
been was
marketed
sometimes
these
tested.
problems,
One of these
synthesized as
an
by Baisse alternative
analog is often combined preparation for both oral
This
preparation
produces
to
with piand par-
little
or no
gastric irritation when given orally, or pain on injection. (2). On the other hand, there is little information on its therapeutic effectiveness, the concentration required in plasma, and its kinetics (2, 3). Before an attempt can be made to gather kiDepartment
versity,
Kingston, Received Sept.
Acid and Theophylline
and Kanji Nakatsu
A spectrophotometric
for
(1970).
(1978)
Spectrophotometry James
B. L., Ion-pair
of Pharmacology, Faculty of Medicine, Ontario K7L 3N6, Canada. 26, 1977; accepted Dec. 1, 1977.
Queen’s
Uni-
netic data it was necessary to develop an assay for theophylline-7-acetic acid because this drug cannot be assayed by the spectrophotometric theophylline assay procedure of Schack and Waxler (4). We describe here a specific spectrophotometric assay in which the serum concentrations of both theophylline-7-acetic acid and theophylline are individually determined. The method requires 2.0 ml of serum and is reliable at concentrations as low as 1 mg/liter.
Materials
and Methods
Equipment. man Model
124UV-Visible
Absorbance
data
were
obtained
spectrophotometer
with
Corp., Maywood, Ill. 60153) scanning from connected to a Recordall Series 5000 chart recorder Scientific, Whitby, Ontario). For dispensing volumes ml or less,
“Pipetman”
(Mandel
Scientific
a Cole-
(Perkin-Elmer 310 to 250
Co.,
Vifie
nm,
(Fisher of 1.0
St. Pierre,
Quebec H8R 1A3) adjustable pipets were used. “Repipet” dispensers (Labindustries, Berkeley, Calif. 94710) were used for volumes greater than 1.0 ml. Chemicals and Reagents. All reagents, reagent-grade unless otherwise noted, were used without pretreatment or special precautions, and can be stored at room temperature, except for citrate buffer, which is stored at 4 #{176}C. The following chemicals were used in preparation of standards and controls; theophylline (as aminophylline), caffeine, theobromine (Sigma Chemical Co., St. Louis, Mo. 63178), theophylline-7-acetic acid (Adams Chemical Co., Round Lake, Ill. 60073), phenobarbital (Allen and Hansburys, Toronto), and 3-methylxanthine (Aldrich Chemical Co., Milwaukee, Wis. 53233). Extraction and analytical procedure. The extraction is done as follows: Pipet a 2.0-mi aliquot of sample or standard CLINICAL
CHEMISTRY,
Vol.
24,
No. 2, 1978
367
was
Table
1. Extraction Efficiency with Use of Various Saturated Salt Solutions RelatIve
efficIency
mining
extraction
of
7-acetic
acId
concentrations,
0.78
Na2SO4 KCI
0.80 0.87
NH4CI
0.89
3-methylxanthine procedure (
