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High-Performance Liquid Chromatographic Determination of

Parecoxib in Human Plasma and Pharmaceutical Formulations

Article  in  Analytical Letters · October 2007

DOI: 10.1080/00032710701606267

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High‐Performance Liquid Chromatographic

Determination of Parecoxib in Human Plasma
and Pharmaceutical Formulations
a a a
S. M. T. Shaikh , D. H. Manjunatha , J. Seetharamappa & P. B. Kandagal
a

a
Department of Chemistry, Karnatak University, Dharwad, India
Version of record first published: 29 Oct 2007.

To cite this article: S. M. T. Shaikh , D. H. Manjunatha , J. Seetharamappa & P. B. Kandagal (2007):

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical
Formulations, Analytical Letters, 40:15, 2925-2934

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 Analytical Letters, 40: 2925–2934, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710701606267

HPLC

High-Performance Liquid
Chromatographic Determination of
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Parecoxib in Human Plasma and

Pharmaceutical Formulations

S. M. T. Shaikh, D. H. Manjunatha, J. Seetharamappa,

and P. B. Kandagal
Department of Chemistry, Karnatak University, Dharwad, India

Abstract: A simple and sensitive RP-HPLC method for the determination of parecoxib
(PXB) in human plasma and pharmaceutical formulations has been developed and
validated. The separation of PXB and the internal standard, ibuprofen (IBF) was
achieved on a CLC C18 (5 m, 25 cm
4.6 mm i.d.) column using UV detector at
200 nm. The mobile phase consisted of acetonitrile-water (92:8 v/v). The linear
range of detection was found to be 0.9– 18.4 mg/ml (r ¼ 0.9985). Intra- and inter-
day assay relative standard deviations were observed to be less than 0.3%. The
method has been applied successfully for the determination of PXB in spiked human
plasma and pharmaceutical preparations. Analytical parameters were calculated and
complete statistical evaluation is incorporated.

Keywords: Parecoxib, human plasma, RP-HPLC

INTRODUCTION

PXB is a prodrug of valdecoxib, a selective inhibitor of cyclooxygenase 2

(COX 2) administered as an intramuscular or intravenous injection (Talley

Received 7 June 2007; accepted 15 July 2007

The authors express their sincere thanks to Glen Copel Marketing, India and Cipla
Ltd., Mumbai, India for providing gift sample of parecoxib and ibuprofen. Thanks are
also due to the authorities of Karnatak University, Dharwad, for providing necessary
facilities.
Address correspondence to J. Seetharamappa, Department of Chemistry, Karnatak
University, Dharwad 580003, India. E-mail: j_seetharam@rediffmail.com

2925
 2926 S. M. T. Shaikh et al.

et al. 2000). Traditionally oral NSAIDs (Barton and Langeland 2002) such as
ibuprofen and diclofenac are used to treat mild to moderate acute pain.
However, this may be problematic when patients are unable to take oral medi-
cation or are nauseous and vomiting. PXB is an effective analgesic in acute
pain (Daniels and Grossman 2001). Critical literature survey revealed that
no attempt has been made so far to develop any analytical method for the
assay of PXB either in bulk, pharmaceutical formulations and plasma
samples. This prompted us to develop a HPLC method for the determination
of PXB in different samples.
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EXPERIMENTAL

Materials

PXB and IBF were obtained as gift samples from Glen Copel Marketing, India
and Cipla Ltd., India, respectively. Acetonitrile (Ranbaxy fine chemicals Ltd.,
Delhi, India) and water (Rankem Ltd., India) used were of HPLC grade.

Stock Solutions

A standard solution of PXB (1000 mg/ml) was prepared in acetonitrile. A

stock solution of IBF (1000 mg/ml) prepared in acetonitrile was used as
internal standard. These stock solutions of PXB and IBF were diluted
further as and when required.

Standard Working Solutions

Working solutions of pure PXB and IBF (500 mg/ml) were prepared separ-
ately in the mobile phase.

Sample Preparation

Blood Plasma

Human blood samples were collected in dry and evacuated tubes (which
contained saline and sodium citrate solution) from different healthy volun-
teers. The samples were handled at room temperature and were centrifuged
for 10 min at 1500 rpm for the separation of plasma within 60 min of collec-
tion. The samples were then transferred to polypropylene tubes and stored at
–208C until analysis.
 High-Performance Liquid Chromatographic Determination 2927

Pharmaceutical Formulation

For injection of PXB, an appropriate volume of the sample was diluted with
mobile phase and then filtered through 0.45 m Millipore membrane filter. It
was then degassed by keeping it in an ultrasonic bath. The known volumes
of these drug solutions were used for the determination.

Operating Conditions
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All HPLC measurements were made on a Shimadzu Corporation system (Analyti-

cal Instruments division, Kyoto, Japan) consisting of a LC10AT solvent pump,
SPD10AVP detector and a data station with win chrome software version 3.1.
The separation was performed on a CLC C18 column (5 m, 25 cm
4.6 mm
i.d.). A CLC ODS (4 cm
4.6 mm, i.d.) was used as a guard column to
protect analytical column. A mixture of acetonitrile and water [92:8, v/v] was
used as a mobile phase at a flow rate of 1.0 ml/min. Hamilton 702 mR injector
with a 25 ml loop was used for the injection of the samples. Measuring at
200 nm effected quantification, and the chromatographic run time was
5 min. The mobile phase was filtered through 0.45 m Millipore membrane
filter and degassed. The separation was carried out at room temperature.

Procedures

Establishment of Calibration Curve

Working solutions of pure PXB (0.9–18.4 mg/ml) containing fixed concen-

tration of internal standard (8 mg/ml) were prepared separately in the mobile
phase. Triplicate 20 ml injections were injected for each working solution to
see the reproducibility of the detector response at each concentration level. A
typical chromatogram obtained is shown in Fig. 1. The peak area ratio of
standard to internal standard was plotted against the concentration of the drug
to obtain the calibration graph. The results were subjected to regression
analysis to calculate calibration equation and correlation coefficient.
Throughout the study, the suitability of the chromatographic system was
monitored by calculating the capacity factor (k0 ), resolution (R), selectivity (a)
and peak asymmetry (AS).

Analysis of Plasma Samples

The plasma samples obtained as described in plasma sample preparation were

allowed to thaw at room temperature before processing. The plasma samples
were spiked with PXB and a fixed amount of IBF (8 mg/ml) and the tube was
briefly shaken. Then the mixture was vortex mixed with ether. The ethereal
 2928 S. M. T. Shaikh et al.
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Figure 1. A typical chromatogram showing the separation of PXB (12 mg/ml) and
IBF (8 mg/ml) in pure form.

layer was evaporated to dryness on a water bath under gentle stream of

nitrogen gas at 408C. The residue was dissolved in suitable amount of
mobile phase and 20 ml solution was injected on to the column.

Analysis of Pharmaceutical Formulation

An aliquot of the drug (obtained by following the procedure described in the

sample preparation for pharmaceutical formulation) was taken and analyzed
using the same chromatographic conditions.

RESULTS AND DISCUSSION

Method Development

The mobile phase was chosen after several trials with methanol, acetonitrile,
isopropyl alcohol, triethylamine, water, and various buffer solutions of
 High-Performance Liquid Chromatographic Determination 2929

different pH in various proportions. Because of high transparency in UV

region, acetonitrile was selected with water as the mobile phase. A mobile
phase consisting of acetonitrile and water (92:8, v/v) was selected to
achieve maximum separation and sensitivity. The effect of flow rate was
investigated by varying the flow rate of the mobile phase from 0.25 to
1.75 ml/min. However, a flow rate of 1.0 ml min21 gave an optimal signal
to noise ratio with a reasonable separation time and hence permitted good
analytical conditions. Various compounds viz., nimesulide, amoxycillin,
piroxicam, cefadroxil, methdilazine hydrochloride, and IBF were examined
as internal standards. A good chromatographic behavior (ideal retention
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time) was observed with IBF and hence, IBF was selected as an internal
standard. Using reverse phase C18 column, the retention times of PXB and
IBF were observed to be 0.82 and 1.74 min, respectively. The total time of
analysis was observed to be less than 5 min. The maximum absorption of
PXB and IBF together were found to be at 200 nm and this wavelength was
chosen for the analysis.

Linearity of Detector Response

Linearity and range of the method were determined by analyzing different

solutions containing 0.1 –20.5 mg/ml of PXB and fixed amount of internal
standard (8 mg/ml) under the chromatographic conditions mentioned
above (n ¼ 9). Then 20 ml solution was injected into the column and chroma-
togram was noted. The peak area ratio of PXB to IBF was plotted against the
concentration of PXB to obtain the calibration graph. Table 1 gives the
regression line, correlation coefficient, slope, intercept and % RSD.
Excellent linearity was noticed in the range of 0.9 – 18.4 mg/ml with
r ¼ 0.9989.

Table 1. Linearity results, limit of detection and limit of

quantification

Parameter PXB

Linear dynamic range (mg/ml) 0.9– 18.4

Regression equation (Y)a —
Slope (b) 0.0892
Interceptw 0.1173
Correlation coefficientw 0.9989
LOD (mg/ml) 0.25
LOQ (mg/ml) 0.85
% RSD 2.11
a
Y ¼ Bx þ c, where X is concentration of drug in mg/ml.
 2930 S. M. T. Shaikh et al.

Limits of Detection and Quantification

Limit of detection (LOD) was established at a signal-to-noise ratio (S/N) of 3

while limit of quantification (LOQ) was calculated at a S/N value of 9. The
LOD and LOQ (Analytical Methods Committee 1987) values were found to
be 0.25 mg/ml and 0.85 mg/ml, respectively (Table 1).

Suitability of the Method

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The suitability of the method was checked by the determination of the chro-
matographic parameters viz., resolution, selectivity and peak asymmetry
and the results are shown in Table 2. The observed values of resolution
(more than 1.5), selectivity (more than 1), and peak asymmetry (less than 2)
revealed ideal chromatographic conditions for the quantification of PXB.

Precision

The precision of the method (Intra- and inter-day variations of replicate deter-
minations) was checked by injecting PXB (n ¼ 9) at the LOQ level. The
results obtained are recorded in Table 3.

Accuracy

A standard working solution containing 15 and 8 mg/ml of PXB and IBF,

respectively, was prepared and the mixture was injected (n ¼ 9) and chroma-
togram was recorded. From the respective area counts, the concentrations of
the PXB were determined using the calibration graph. The accuracy,
defined in terms of % deviation of the obtained concentration from the
actual concentration is listed in Table 3.

Table 2. System performance parameters of PXB and IBF

Parameters PXB IBF

Retention time (tR) in min 0.82 1.74

Capacity factor (k0 ) 1.73 4.8
Selectivity factor (a) 2.7
Resolutionw 1.80
Peak asymmetry (AS) 1.11 1.24
Number of theoretical plates (N) 15.02 56.86
Height equivalent to theoretical 99.86 26.38
plate (H ) in mm
 High-Performance Liquid Chromatographic Determination
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Table 3. Intra-day (1 representative day) and inter-day precision and accuracy for the determination of PXB

Intra-day Inter-day
(n ¼ 9) (n ¼ 9)
Conc. added, concentration concentration
mg/ml measured % RSD % Bias measured % RSD % Bias

5 4.91 0.285 21.80 4.89 0.210 22.2

10 9.82 0.212 21.81 9.79 0.186 22.1
15 14.81 0.224 21.26 14.75 0.206 21.7

2931
 2932 S. M. T. Shaikh et al.

Ruggedness

The ruggedness of the proposed method was evaluated by carrying out the
analysis of the working solution using the same chromatographic system
and the same column on different days (Nagaralli et al. 2003). Small
differences in area-ratios and good constancy in retention times were
observed after 48 h time period. The RSD values of less than 1.5% for both
areas and retention times were obtained (Table 4). The comparable detector
responses obtained on different days indicated that the method is capable of
producing results with high precision on different days.
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The ruggedness of the method was examined by injecting the standard

working solution into a different HPLC unit. The high degree of reproducibil-
ity of the detector response and retention times indicates that the method is
fairly rugged.

Specificity of the Method

The specificity of the proposed method was confirmed by the fact that the
drugs viz., nimesulide, amoxycillin, cloxacillin, cefadroxil, methdilazine
hydrochloride, diclofenac sodium, and ciproflaxacin did not interfere in the
determination, as evident from their retention times which are different
from that of PXB.

Applications

Analysis of Plasma Samples

The proposed method was applied to the determination of PXB in spiked

plasma samples. The results obtained for precision and accuracy at the three
different concentrations in plasma are recorded in Table 5. Low values of

Table 4. Day to day variability according to area and

retention time

1st Day 2nd Day

Areaa 10208777 10208824

SD 127609 147007
% RSD 1.25 1.44
Retention timea 0.82 0.83
SD 0.0045 0.0052
% RSD 0.55 0.63
a
Mean values of nine determinations.
 High-Performance Liquid Chromatographic Determination 2933

Table 5. Precision and accuracy data for the determination of PXB in human plasma
samples

Measured
Concentration concentration,a
Drug added mg/ml mg/ml % RSD % Bias

PXB 5 4.91 + 0.09 1.8 21.8

10 9.80 + 0.23 2.3 22.0
15 14.75 + 0.37 2.5 21.6
a
Average of five determinations.
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relative standard deviation indicate high precision of the proposed method.

The proposed method is found to be accurate as evident from low % Bias
values.

Analysis of Pharmaceutical Preparation

The proposed method was successfully applied to the analysis of PXB in injec-
tions and the results are shown in Table 6. The low values of relative standard
deviation indicate high precision of the method.

Recovery Studies

To study the accuracy and reproducibility of the proposed method, recovery

experiments were carried out. The recovery of the added standard was
studied at five different levels. Each level was repeated five times. To
aliquots of the previously analyzed preparations, a known concentration of
standard solution of PXB was added. The content of PXB was once again
determined by the proposed method. From the amount of drug present,

Table 6. Analysis of PXB in pharmaceutical formulations

Labeled, Found,b
a
Injection mg/ml mg/ml % RSD % Recovery

Vorth-P 40 39.6 + 0.56 1.41 99

Valus-P 40 40.4 + 0.44 1.08 101
a
Marketed by Glen Copel Marketing.
b
Average of five determinations.
 2934 S. M. T. Shaikh et al.

percentage recovery was calculated using the following formula:

P 
P 
P 
N xy  y x
% Recovery ¼
P 
P 2
N x 2 x
where, x ¼ amount of standard drug added, y ¼ amount of drug found by
proposed method, N ¼ number of observations.
The results were found to be in the range of 97.8% to 98.4%.

CONCLUSION
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The proposed HPLC method showed acceptable linearity, precision and

accuracy over the concentration range mentioned. The method described is
rapid, since preparation of plasma samples prior to chromatography is
relatively simple and the total chromatographic run time is about 5 min.
The chromatographic method could be used to analyze a large number of
plasma samples each day in clinical laboratories and pharmaceutical formu-
lations in quality control laboratories. Hence, the proposed method could be
adopted for the assay of PXB in plasma samples and pharmaceutical
preparations.

REFERENCES

Analytical Methods Committee. 1987. Recommendations for the detection, estimation,

and use of the detection limit. Analyst, 112: 199.
Barton, S.F. and Langeland, F.F. 2002. Efficacy and safety of intravenous parecoxib
sodium in relieving acute postoperative pain following gynecologic laparotomy
surgery. Anesthesio., 97: 306.
Daniels, S.E. and Grossman, E.H. 2001. COX II inhibitors in acute analgesia. Clin.
Ther., 23: 1018.
Nagaralli, B.S., Seetharamappa, J., Gowda, B.G., and Melwanki, M.B. 2003. Liquid
chromatographic determination of ceterizine hydrochloride and paracetamol in
human plasma and pharmaceutical formulations. J. Chromatogr. B., 798: 49.
Talley, J.J., Bertenshaw, S.R., Brown, D.L., Carter, J.S., Graneto, M.J., Kellogg, M.S.,
Coboldt, C.M., Yuan, J., Zhang, Y.Y., and Seibert, K. 2000. 4-[5-Methyl-3-pheny-
lisoxazol-4-yl]-benzenesulfonamide, Valdecoxib: A Potent and Selective Inhibitor
of COX-2. J. Med. Chem., 43: 775.

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