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The following message was posted to: PharmPK
Hello everybody, Happy New Year to every one..
i would like to know spectrophotometric (UV-visible) method of
of nimesulide and references for pH-solubility studies of nimesulide.
expecting a quick reply..
* SK. Abdul Mohammed Jafar Sadik Basha *
* 2002H108032 *
* Room No.: 230, Bhagirath Bhavan *
* BITS, Pilani *
* Rajasthan *
* Phone No.: 0159-7642809 *
* E-mail id: h2002032.-at-.bits-pilani.ac.in*
* jafar_lp.-at-.yahoo.com *
* bas_sadik.aaa.rediffmail.com *
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Enclosed is an HPLC-UV assay for nimsulide.
Clin Drug Invest 21(5):361-369, 2001. © 2001 Adis International Limited
Study of Two Different Nimesulide-Containing Preparations
from Clinical Drug Investigation [TM]
Materials and Methods
Eighteen healthy male Caucasian volunteers were enrolled in the study.
Demographic data and other baseline characteristics are reported in
After having been informed by the medical supervisor of the trial about
aim, course and possible risks of this investigation, all volunteers
written consent for their participation. The participants were selected
the basis of acceptable medical histories and normal physical
which did not show any clinically significant hepatic, renal, cardiac,
gastrointestinal or haematological deviations or any acute or chronic
A comprehensive laboratory screening was performed within 21 days
start of the study to confirm the good state of health of the
Further inclusion criteria were a negative drug screening, a negative
alcohol test and a negative HIV and hepatitis B antigen screening.
days after completion of the study, a detailed medical examination of
participants was carried out and clinical chemistry parameters were
The protocol was approved by an Institutional Review Board to be in
accordance with the Declaration of Helsinki in its revised Somerset West
edition (October 1996), the German
Arzneimittelgesetz and the recommendations of the US Food and Drug
Administration. The study was conducted with full Good Clinical
Good Laboratory Practice compliance.
Both tested formulations were commercially available in Italy. The test
preparation was the original product Aulin® (Boehringer Mannheim, Italy)
100mg tablets (batch no. 276), while the reference drug preparation was
generic nimesulide-containing preparation, Nimesulide Dorom 100mg
(batch no. 1123A) [Dorom s.r.l., Italy].
The study was performed as a single-dose, randomised, two-way crossover
design in 18 healthy volunteers. Randomisation was performed in blocks
two participants each. Participants as well as investigators could be
of the preparations administered during each period, whereas the
allocation was blinded for the analyst.
The two study periods were identically set up. Between the two
administrations a wash-out period of 7 days was observed. Participants
admitted to the clinical centre around 4 to 6pm on the afternoon prior
drug administration. They remained in the clinic until collection of the
last blood sample during each run. During hospitalisation, participants
provided with standardised meals. The consumption of alcohol or
not allowed during this time.
Drug administration on medication days took place at about 8am after an
overnight fast. Both preparations were administered with approximately
of water by a clinical staff member. The complete and correct intake of
preparations was subsequently checked by means of a thorough inspection
the oral cavity with the aid of a flashlight. No food was permitted
hours after drug administration.
Blood samples (9ml of venous blood) were collected at 0 (predose), 20
40 min, 1h, 1h 20 min, 1h 40 min, 2h, 2h 30 min, 3h, 3h 30 min, 4h, 5h,
40 min, 9h, 12, 16h and 24h postadministration.
Directly after blood withdrawal, the blood samples were centrifuged and
plasma was transferred into polystyrene tubes and stored frozen at -20°C
The concentrations of nimesulide and its main metabolite
4'-hydroxy-nimesulide in the plasma samples were determined through a
validated analytical method using high performance liquid chromatography
(HPLC) in combination with a UV detection technique. The method,
in the laboratories of AAI, allows simultaneous determination of
and the metabolite. In detail, the isocratic liquid chromatography was
performed with a mobile phase consisting of 1000g methanol, 650g
acetonitrile, 1650g water, 10g disodiumhydrogenphosphate, and 4g heptan
sulfonic acid, and the pH was adjusted to 5.5 by using phosphoric acid
(85%). The mobile phase was delivered at a flow-rate of 0.7 ml/min and
separation was accomplished at 35°C on a Supersphere Select B, 5µM, 125
3mm internal diameter. The UV-detector was set at 297nm.
Nimesulide and the metabolite were extracted from plasma samples and
analysed in defined runs. Extraction was performed as follows: a 0.5ml
aliquot of the plasma sample was mixed with 100µl internal standard (100
mg/ml), 400µl sodium-tetraborate buffer (0.01 mol/L) and 6ml chloroform,
respectively. After overhead shaking and centrifugation for 20 minutes,
organic solvent was evaporated to dryness under a stream of ntirogen at
50°C. The residue was redissolved in mobile phase and an aliquot was
injected into an HPLC system. One analytical run (covering one sample
consisted of 34 individual samples (all samples of both periods of one
individual), a calibration curve with eight different concentrations, a
control blank and two sets of quality control samples (spiked plasma
containing 0.08, 0.80 and 4 mg/L of nimesulide and
respectively) [low-, medium-and high-concentration controls].
The calibration range for nimesulide and metabolite extended from 0.05
5µg with calibration standards (n = 8) being set at concentrations of
0.075, 0.10, 0.25, 0.50, 1, 2.5 and 5 mg/L of nimesulide and
4'-hydroxy-nimesulide. The lower (LOQ) and upper (ULQ) limits of
quantification were 0.05 and 5.0 mg/L for both the parent compound and
The in-process assay of quality control samples (n = 36) spiked with
concentrations of 0.08, 0.80 and 4 mg/L resulted in coefficients of
variation of 2.9, 1.8 and 1.4%, respectively, for nimesulide and 3.1,
and 1.7% for the metabolite.
Pharmacokinetic Calculations and Statistics
The areas under the plasma concentration-time curves (AUCs) of
and 4'-hydroxy-nimesulide were calculated according to the trapezoidal
In this calculation, AUC0-z represents the area under the plasma
concentration-time curve from the time of drug administration (time 0)
the last sample with a quantifiable concentration, AUC0-24 indicates the
area from time 0 to the last scheduled sample at 24 hours after
administration, and AUC0-infinity is the corresponding area
infinity. The values for Cmax and tmax were taken directly from the
t1/2z was determined by log-linear regression of the elimination phase.
addition, for nimesulide the ratio Cmax/AUC0-infinity was calculated.
AUC0-infinity/AUC0-infinity were compared by analysis of variance, ANOVA
[(utilising general linear models (GLM)] after logarithmic
transformation evaluating for sequence, participant-within-sequence,
period and treatment effects. 90% confidence intervals were calculated;
values were compared by Koch's nonparametric analysis procedure together
with derivation of the nonparametric confidence interval according to
All tests and comparisons were evaluated at the 95% significance level
I hope it helps.
Louis R. Ptak, Ph.D.
Senior Medical Writer
ICON Clinical Research, Inc.
212 Church Road
North Wales, Pennsylvania 19454
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The following message was posted to: PharmPK
Dear Mr. Basha
Being a weak acid, nimesulide has a very low solubility in an acidic pH
and the pH-solubility profile of nimesulide is such that it increases
with an increase in the pH of the system which favours ionisation of
the molecule. An increase in the pH of the system, results in
ionisation of the molecule with the appearance of a yellow colour with
a lambda max of about 389 nm and can be used for its estimation.
Manish Chawla, Ph.D.
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