406
M. Mokrý et al./Chemical Papers 64 (3) 405–408 (2010)
I. D. column packed with Discovery C18 (5 µm par-
ticle size) (Sigma–Aldrich, Prague, Czech Republic).
The mobile phase consisted of a mixture of methanol
and 0.01 M solution of dibasic ammonium phosphate
(ϕr = 60 : 40) whose pH was adjusted to 4.0. The flow
rate was 1.0 mL min−1 at room temperature (24◦C)
and the detector wavelength was set to 254 nm. Injec-
tion was carried out by means of a loop of the volume
of 20 µL.
Nimesulide, methylparaben, propylparaben, 4-hyd-
roxybenzoic acid, and phenacetine were purchased
from Sigma–Aldrich (Prague, Czech Republic). All
solvents used were of HPLC purity grade; diba-
sic ammonium phosphate (Merck, Říčany-Jažlovice,
Czech Republic) and other chemicals were of ana-
lytical grade. 2-Phenoxy-4-nitroaniline was obtained
by alkaline hydrolysis of nimesulide. The crystals ob-
tained by acidification (pH = 3) of the alkaline solu-
tion were filtered off, recrystallized from a mixture of
methanol/water (ϕr = 1 : 1) and their structure was
confirmed by NMR.
Fig. 1. Chromatogram of the analysed syrup after hydrolysis
at 90◦C for 2 h: 4-HBA (1), MP (2), PAC (3), PP (4),
NIM (5), PNA (6).
The stock solution of NIM was prepared by weigh-
ing of about 25 mg of NIM accurately, the weighed
amount was dissolved in methanol and diluted with
it in a volumetric flask to the volume of 25 mL. The
solution of the internal standard (phenacetine, PAC)
in methanol was prepared in such a way as to obtain
its resultant concentration of 0.5 mg mL−1. For the
calibration curve, NIM solutions of the con−ce1ntrations
of 50 µg mL−1, 70 µg mL−1, 100 µg mL , 120 µg
mL−1, and 150 µg mL−1 with 50 µg mL−1 of the
internal standard were prepared. Stock solutions of
PNA, MP, PP, and HBA (all 1 mg mL−1) were pre-
pared in methanol. All solutions were stable during
the described chromatographic analysis.
For optimization, the sample solutions were pre-
pared diluting the analytes in the mobile phase to the
concentrations of 0.2 mg mL−1 of NIM and phenace-
tine and to 0.1 mg mL−1 of PNA, MP, PP, or HBA.
The syrup containing 10 mg mL−1 of NIM was di-
luted with the mobile phase to concentration of 100
µg mL−1. The amount of the internal standard solu-
tion added was 50 µg mL−1, and the content of NIM
was assayed. For the evaluation of impurities, 1 mL of
the syrup was diluted to 5 mL. To prepare samples for
accuracy and precision measurements, the stock solu-
tions were diluted with the mobile phase. Finally, as
the method was proposed not only for impurity evalu-
ation but also for stability assays, forced degradation
was performed. For this process, 1 mL of the syrup
was diluted to 10 mL by 1 M potassium hydroxide
and it was heated at 90◦C for 2 h. After the dilution,
the sample was injected into the column.
NHSO2CH3
O
NH2
NO2
O
CH SO K
+
3
3
NO2
NIM
PNA
Fig. 2. Alkaline hydrolysis of NIM yielding PNA and potas-
sium methanesulphonate.
ionisation of acidic SiOH sites on the silica gel sur-
face resulted in a complete separation of NIM, PNA,
PAC, MP, PP, and HBA. At higher values of pH, the
separation of HBA and MP was not complete. The
optimisation was continued by changing the amount
of methanol. A chromatogram showing the resolution
is given in Fig. 1.
The degradation product, the retention time of
which is about 14 min in HPLC chromatographic
records, was isolated from a hydrolysate heated at
100◦C under a reflux condenser for 50 h. Scheme of
the alkaline hydrolysis of NIM is given in Fig. 2.
Calibration curves for the determination of the
evaluated substances were generated by the least
squares linear regression of the peak area ratios (y)
vs. concentrations (x). For the unknown samples, the
peak area ratios were calculated from the integrated
peaks of the chromatograms; and then, they were con-
verted to concentrations using the linear regression
parameters (slope and intercept). The HPLC method
was tested for linearity, precision and accuracy. Lin-
earity of the method was determined by preparing
and analysing a series of five standard solutions in
the range presented in Table 1. The results from the
method validation are listed in Table 2. The method
A water/acetonitrile (ϕr = 50 : 50) chromato-
graphic system without pH control gave limited speci-
ficity and reproducibility. Changing the organic mod-
ifier to methanol and the aqueous phase to dibasic
ammonium phosphate buffer of pH 4.0 to reduce the
Mokry, Milan
