GUERMOUCHE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO.

6, 2000 1489
SPECIAL GUEST EDITOR SECTION

Assay of Naproxen in Rat Serum by High-Performance

Thin-Layer Chromatography/Densitometry
MOULAY-HASSANE GUERMOUCHE and NADIA ATIK
UniversitJ des Sciences et Technologies Houari Boumedienne, Institut de Chimie, Centre de Recherche en Analyse
Physico-Chimique, BP No. 32, El-Alia, Bab-Ezzouar, Alger, Algeria
HOCINE CHADER
Institut National des Etudes en Sciences MJdicales, Departement de Pharmacie, Laboratoire National de Contr^le, Alger,
Algeria

A simple, sensitive, and reproducible

high-performance thin-layer chromatographic
method using densitometry is presented for the
determination of naproxen in rat serum. Only
0.1 mL serum was used for extraction. Separations were performed on 10 10 cm plates coated
with silica gel, with tolueneethyl acetateacetic
acid (82 + 15 + 3) as the mobile phase. Benzophenone was used as the internal standard. Quantification was performed by densitometry at 260 nm.
The response for naproxen was linear (r = 0.992)
over the range 2100 mg/L. Method validation
demonstrated good recoveries (9296%), sensitivity (limit of quantitation, 1 mg/L), repeatability
of sample application (4%), repeatability of the
method (8%), and intermediate precision (5%).
The procedure was applied to the quantitation of
naproxen in rat serum.

aproxen, (S)-6-methoxy-"-methyl-2-naphthaleneacetic acid, is used in painful osteoarthritis and rheumatoid arthritis. Activity resides mainly in the S-enantiomer. Naproxen inhibits cyclooxygenase and decreases prostaglandin concentrations in fluids and tissues. Some side effects
have been described, such as gastrointestinal bleeding and reduction of kidney function (1). Therapeutic activity based on
clinical indexes is established with a daily dose of
5001500 mg (2). Naproxen is primarily metabolized to
6-O-demethylnaproxen, and both compounds are conjugated
to their glucuronides.
To determine the plasma concentration of naproxen, a
number of analytical techniques are described in the literature
including spectrofluorimetry (35), gas chromatography
(614), and recently spectrophotometry (15). Actually, liquid
chromatographic (LC) methods seem to be more convenient.
A wide variety of reversed-phase LC methods with various
sensitivities have been reported for the analysis of small

Guest edited as a special report on Thin-Layer Chromatography/

Densitometry by Joseph Sherma.

plasma samples (1621). Recently, electrokinetic capillary

chromatography was also used successfully (22). These procedures used sample pretreatments such as solvent extraction,
protein precipitation, or solid-phase extraction. Thin-layer
chromatography (TLC) was used less frequently (2325), and
the methods described in these references combined TLC with
spectrofluorimetry. They are tedious and time consuming and
require external standardization. High-performance (HP)
TLC was not applied to the determination of naproxen in
plasma; however, it was used for the determination of
ibuprofen (26) and flurbiprofen (27).
In this study, we describe a simple, rapid, sensitive, and
specific HPTLC method for the determination of naproxen in
serum. Compared with the LC methods, the HPTLC assay is
more attractive because of its speed and simplicity. HPTLC
facilitates quantitation by scanning and analysis of several
samples simultaneously. The proposed method features
naproxen extraction with a small serum volume and
densitometric quantitation using an internal standard.
Experimental

Reagents
All solvents (spectroscopic grade) used were from Merck
(Manheim, Germany). Naproxen and benzophenone, which
were used as the internal standard, were from Sigma (St.
Louis, MO).

Apparatus
Chromatography was performed on 10 10 cm HPTLC
plates precoated with Silica Gel 60, 200 m layer thickness
(Merck, Darmstadt, Germany). Samples were applied to the
plate with a Camag (Muttenz, Switzerland) microapplicator,
8 mm from the lower edge of the plate and 10 mm apart, starting 10 mm from the side of the plate. Development was
performed in a linear developing chamber (Camag). Zones
were quantitated by densitometry with a TLC/HPTLC Scanner III and CATS 4 software (Camag) for a distance of 8 cm at
ambient temperature by using a measurement wavelength of
260 nm and slit dimensions of 5 0.1 mm. Each plate was
scanned 3 times. Peak areas were used for quantitation. Average quantitative results were given. Scanning speed was kept

1490 GUERMOUCHE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 6, 2000

at 1 mm/s. Plates were developed with several mobile phases

containing toluene, ethyl acetate, and acetic acid.

Sample Preparation
Serum samples were collected from Wistar rats that received naproxen intraperitonially at 8 mg/kg. Blood samples
were collected from the orbital venous plexus. Serum samples
were separated by centrifugation at 6000 rpm for 15 min. To
improve sample stability, they were stored at 20C and allowed to defrost at 25C before use. Each serum sample was
analyzed 10 times.
To 0.1 mL serum were added 0.01 mL of a 500 mg/L solution of benzophenone (internal standard) in methanol and
0.02 mL 1M HCl. Extraction was performed with 2 mL diethyl
ether. The organic layer was transferred to a clean test tube and
evaporated to dryness. The dry residue was reconstituted with
0.1 mL mobile phase, and 1 L was spotted on the plate.
Spotting >1 L on the plate saturates the silica gel and introduces tailing that affects peak area and resolution.

Calibration
Calibration was accomplished by using the following procedure. To ten 0.08 mL samples of blank serum were added
0.01 mL of various concentrations of naproxen
(201000 mg/L) and 0.01 mL of a constant internal standard
concentration (500 mg/L). In the samples obtained, the
naproxen concentration varied from 2 to 100 mg/L, and the internal standard concentration was fixed at 50 mg/L. The samples were extracted and analyzed according to the procedure
described above to establish the calibration curve in the presence of serum. The use of an internal standard limits the errors
occurring during the naproxen extraction. The advantages of
this calibration method are that the naproxen quantities injected need not be accurately measured because the detector
responses do not alter the area ratio.
The calibration curve in the absence of serum was established by analyzing standard solutions in which the naproxen
concentration varied from 2 to 100 mg/L, and the internal standard concentration was fixed at 50 mg/L. The solutions were
obtained as follows. To ten 0.08 mL portions of methanol were
added 0.01 mL of various concentrations of naproxen
(201000 mg/L) and 0.01 mL of a constant internal standard
concentration (500 mg/L). In the solutions obtained, the
naproxen concentration varied from 2 to 100 mg/L, and the internal standard concentration was fixed at 50 mg/L. The solutions were analyzed according to the procedure described
above to establish the calibration curve in the absence of serum.

Precision of Sample Applications

The within-run precision of sample applications was estimated from 10 analyses of the same aliquot of blank serum
containing added naproxen and internal standard. To 0.08 mL
serum were added 0.01 mL of a constant concentration of
naproxen (500 mg/L) and 0.01 mL internal standard in methanol at 500 mg/L. The sample was extracted and spotted
10 times on the same plate.

Repeatability
Repeatability was determined by analyzing ten 0.8 mL portions of blank serum containing added naproxen and internal
standard. To each 0.8 mL blank serum was added 0.1 mL of a
constant concentration of naproxen (500 mg/L) and 0.1 mL
internal standard at 500 mg/L. The 10 samples were extracted
and spotted on the same plate.
Results and Discussion

Mobile Phase Selection

HPTLC experiments were conducted with mobile phases
containing ethyl acetate, toluene, and acetic acid in which the
amounts of toluene and ethyl acetate were changed. The variation of the retention factors of the internal standard and
naproxen with the ethyl acetate content of the mobile phase is
shown in Figure 1. Below 10% ethyl acetate, the retention factor, RF, of naproxen is too low; its corresponding peak could
interfere with the endogenous compounds present in serum
and appearing with a small RF. Above 30% ethyl acetate, the
RF of the internal standard is near one. The choice of the best
chromatographic conditions was a compromise that depended
on the retention factors of naproxen and benzophenone. It appeared that the best separation between naproxen and the internal standard occurred with tolueneethyl acetateacetic
acid (82 + 15 + 3). The retention factors of naproxen and benzophenone obtained with this mobile phase were 0.31 and
0.73, respectively. Retention factors obtained for some other
anti-inflammatory drugs with the proposed HPTLC method

Detection Limit
The detection limit was determined at a signal-to-noise ratio of 4:1.

Recovery
At a specific concentration, recovery was determined from
the ratio of the calibration curves in the presence and in the absence of serum.

Figure 1. Variation of the retention factors of naproxen

and the internal standard with the ethyl acetate content
of the mobile phase.

GUERMOUCHE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 6, 2000 1491

Figure 2. Typical chromatograms (wavelength = 260 nm) of (a) blank serum and (c) rat serum (dose: 8 mg/kg
intraperitonially; time of serum collection: 1 h 30 s; (AS MEANT?) naproxen serum concentration: 70 mg/L).
Chromatogram (b) corresponds to the blank serum measured at 230 nm.

were 0.10 for flurbiprofen, 0.17 for ibuprofen, 0.40 for

indoprofen, and 0.47 for ketoprofen. Naproxen was clearly
separated from the other drugs. We did not study the possible
interferences of naproxen metabolites because in plasma only
naproxen could be detected (20). The proposed mobile phase
is different from the pharmacopeial mobile phase (28), toluenetetrahydrofuranacetic acid (30 + 3 + 1), which is more
suited to the determination of the degradation products in raw
material.

HPTLC Results
The therapeutic range of naproxen in serum is between
5 and 50 mg/L. The calibration graph was obtained for the
range 2100 mg/L. The least-squares regression equation of
the calibration curve was linear. Statistical treatment of the
calibration curves in the presence or absence of serum was estimated through the slope and intercept relative errors. Data
for the calibration curve in the absence of serum were as follows: slope, 42 104; relative error of slope, 2%; intercept,
9.6 104; relative error of intercept, 6%; and correlation coefficient, 0.996. Data for the calibration curve in the presence
of serum were as follows: slope, 44.7 104; relative error of
slope, 4%; intercept, 11.7 104; relative error of intercept,
8%; and correlation coefficient, 0.992.
The detection limit of the assay (determined with a baseline signal-to-noise ratio of 4:1) in the proposed method is
1 mg/L. A lower detection limit can be found at a lower UV
wavelength (for example, at 230 nm), but endogeneous interferences in the naproxen determination may occur. Indeed, at

a retention factor of 0.31, which corresponds to that of

naproxen, the 230 nm baseline absorbance of the blank serum
(labeled naproxen 230 nm in Figure 2b) extract is high when
compared with the corresponding 260 nm baseline
absorbance (labeled naproxen 260 nm in Figure 2b).
Recovery was determined from the ratio of the calibration
curves in the presence and the absence of serum. The values
found were satisfactory over the entire therapeutic range. For
the lowest naproxen concentrations (15 mg/L), recoveries
were 9294%; for naproxen concentrations of 550 mg/L, recoveries were 9496%.
The other results, determined as described in the Experimental section, reveal the good repeatability of sample application (4%), repeatability of the method (8%), and intermediate precision (5%).

Table 1. Determination of naproxen in rat serum by the

proposed HPTLC method
Rat

Time of serum collection,

h after dosing

Naproxen
found, mg/La

Relative standard
deviation, %

1.5

70

2.9

81

3.1

83

3.6

77

3.6

Each value is the average of 10 determinations.

1492 GUERMOUCHE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 6, 2000

Rat serum samples were analyzed by the proposed

HPTLC method. A typical chromatogram is shown in Figure 2c. No interfering peaks were noticeable (see
chromatogram of blank serum in Figure 2a). The quantitative
results are given in Table 1.
Conclusions
The HPTLC method enables quantitation of naproxen in
rat serum in the concentration range of 2100 mg/L. It includes solvent extraction after addition of an internal standard,
evaporation to dryness, residue dissolution, and densitometric
measurement after chromatographic separation. The repeatability of sample application, the repeatability of the method,
the intermediate precision, the linearity, and the recoveries by
the proposed method are excellent. Only 0.1 mL serum is
needed for analysis. The limit of detection was found to be
1 mg/L when the therapeutic range of naproxen in serum was
between 5 and 50 mg/L. The assay described is attractive because of its speed, simplicity of sample treatment, and the
large number of samples that can be analyzed simultaneously
with a small quantity of solvent. It can be used for routine
analysis in the clinical laboratory after its adaptation to samples of human serum.
References
(1) Todd, P.A., & Crissold, S.P. (1990) Drugs 40, 9195
(2) Verbeek, R.H., Blackburn, J.L., & Loewen, G.R. (1983) Clin.
Pharmacokinet. 8, 297301
(3) Antilla, M. (1977) J. Pharm. Sci. 66, 433434
(4) Antilla, M., Hattaaja, M., & Kasanen, A. (1980) Eur. J. Clin.
Pharmacol. 18, 263268
(5) Mortensen, A., Bjorn, J.E., Petersen, P.B., Husted, S., &
Andreasen, F. (1979) Acta Pharmacol. Toxicol. 44, 277283
(6) Runkel, R., Chaplin, M., Boost, G., Segre, E., & Forchielli,
E. (1972) J. Pharm. Sci. 61, 703708
(7) Runkel, R., Krafts, S., Boost, G., Sevelius, H.E., Forchielli,
E., Hill, R., Magoun, R., Szakas, J.B., & Segre, E. (1972)
Chem. Pharm. Bull. 20, 14571466

(8) Runkel, R., Forchielli, E., Boost, G., Chaplin, M., Hill, R.,
Sevelius, H., Thompson, G., & Segre, E. (1973) Scand. J.
Rheumatol. 2, 2935
(9) Runkel, R., Forchielli, E., Sevelius, H., Chaplin, G., & Segre,
E. (1974) Clin. Pharmacol. Ther. 15, 261266
(10) Runkel, R., Chaplin, R., Sevelius, H., Ortega, E.G., & Segre,
E. (1976) Clin. Pharmacol. Ther. 20, 269277
(11) Runkel, R., Mroszcak, E., Chaplin, R., Sevelius, H.G., &
Segre, E. (1978) Clin. Pharmacol. Ther. 24, 706713
(12) Day, R.O., Furst, D.E., Dromgoole, S.H., Kamm, B., Roe, R.,
& Paulus, H.E. (1982) Clin. Pharmacol. Ther. 31, 733737
(13) Sevelius, H., Runkel, R., Segre, E., & Bloomfield, S.S.,
(1980) Br. J. Clin. Pharmacol. 10, 259265
(14) Weber, S.S., Bankhurst, A.D., Mroszcak, E., & Ding, T.L.
(1981) Ther. Drug Monit. 3, 7583
(15) Khan, I.U., Aman, T., Ashraf, A., & Kagi, A.A. (1999) Anal.
Lett. 32, 20352039
(16) Tsai, Y.H., Hsu, L.R., & Naito, S.J. (1985) Int. J. Pharm. 24,
101106
(17) Heizman, P., Korner, J., & Zinapold, K. (1986) J. Chromatogr.
374, 95102
(18) Richardson, C.J., Ross, S.G., Blocka, K.L., & Verbeeck, R.K.
(1986) J. Chromatogr. 382, 382389
(19) Blagbrough, I.S., Daykin, M.M., Doherty, M., Pattrick, M., &
Shaw, P.N. (1992) J. Chromatogr. 578, 251257
(20) Vree, T.B., Biggelaar-Martea, M., & Verwey-van Wissen,
C.P.W.G.M. (1992) J. Chromatogr. 578, 239249
(21) Satterwhite, J.H., & Boudinot, F.D. (1988) J. Chromatogr.
431, 444449
(22) Albrecht, C., & Thormenn, N. (1998) J. Chromatogr. 802,
115122
(23) Lotti, B. (1975) Boll. Chim. Farm. 114, 351354
(24) Ballerini, R., Cambi, A., & Del Soldato, P. (1977) J. Pharm.
Sci. 66, 281282
(25) Abdel-Moety, E.M., Al-Obaid, A.M., Jado, A.I., & Lofti, E.A.
(1988) Eur. J. Drug Metab. Pharmacokinet. 13, 267271
(26) Save, T.K., Parmar, D.V., & Devarajan, P.V. (1997)
J. Chromatogr. 690, 315319
(27) Dhavse, V.V., Parmar, D.V., & Devarajan, P.V. (1997)
J. Chromatogr. 694, 449453
(28) Pharmacope Europenne (1997) 3rd Ed., European Directorate for the Quality of Medicines, Strasbourg, France,
p. 1244