A simple, rapid, precise, accurate, reproducible, economic and eco-friendly analytical method for the quantification
of piroxicam in tablet dosage form, using attenuated total reflectance Fourier transform infrared spectroscopy (ATRFTIR), has been developed. The measurements were based on the ATR technique, wherein there is no sample
preparation, except for pulverizing the tablet, thereby eliminating the use of toxic chemicals or solvents. The calibration
curve of piroxicam was obtained by performing a linear regression analysis, using data from the direct ATR-FTIR
measurement of percentage transmittance (%T), corresponding to the secondary peak at 1526.38 cm-1 in the
concentration (C) range of 25.18-93.63 mg/g. The linear regression equation obtained from the present data was
represented by %T = -0.2178 C + 99.208 and was associated with a coefficient of determination (r2) of 0.998. The
proposed method was validated for piroxicam in tablet form. The relative standard deviation percentage (%RSD) was
found to be less than five, with a recovery percentage in the range of 96.0-99.3 %.
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Cite this paper: Vietnam J. Chem., 2020, 58(5), 615-621 Article
DOI: 10.1002/vjch.202000049
615 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH
Attenuated total reflection-Fourier transform infrared
spectroscopy (ATR-FTIR) for quantification of piroxicam in tablet
dosage from as a green analytical method
Nguyen Quoc Thang1*, Nguyen Thi Mai Tho1, Ho Ngoc Nga1, Nguyen Thi Kim Phuong2,3*
1Chemical Engineering Faculty, Industrial University of Ho Chi Minh City,
12 Nguyen Van Bao, Go Vap district, Ho Chi Minh City 70000, Viet Nam
2Institute of Resources Geography, Vietnam Academy of Science and Technology,
01 Mac Dinh Chi, district 1, Ho Chi Minh City 70000, Viet Nam
3Graduate University of Science and Technology, Vietnam Academy of Science and Technology,
18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam
Submitted March 30, 2020; Accepted May 20, 2020
Abstract
A simple, rapid, precise, accurate, reproducible, economic and eco-friendly analytical method for the quantification
of piroxicam in tablet dosage form, using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-
FTIR), has been developed. The measurements were based on the ATR technique, wherein there is no sample
preparation, except for pulverizing the tablet, thereby eliminating the use of toxic chemicals or solvents. The calibration
curve of piroxicam was obtained by performing a linear regression analysis, using data from the direct ATR-FTIR
measurement of percentage transmittance (%T), corresponding to the secondary peak at 1526.38 cm-1 in the
concentration (C) range of 25.18-93.63 mg/g. The linear regression equation obtained from the present data was
represented by %T = -0.2178 C + 99.208 and was associated with a coefficient of determination (r2) of 0.998. The
proposed method was validated for piroxicam in tablet form. The relative standard deviation percentage (%RSD) was
found to be less than five, with a recovery percentage in the range of 96.0-99.3 %.
Keywords. Piroxicam, ATR-FTIR, tablet dosage.
1. INTRODUCTION
The chemical structure of piroxicam (IUPAC name:
4-hydroxy-2-methyl-1,1-dioxo-N-pyridin-2-yl-1λ6,
2-benzothiazine-3-carboxamide) is shown in figure
1. Piroxicam is used to reduce pain, swelling,
and joint stiffness due to arthritis. It is a nonsteroidal
anti-inflammatory drug (NSAID). Piroxicam acts by
blocking the production of certain natural substances
that cause inflammation in the human body.[1,2]
Figure 1: Chemical structure of piroxicam
Several methods have been used for the
quantification of piroxicam in pharmaceutical
formulations, such as spectrophotometry, high-
performance liquid chromatography, voltammetry,
differential pulse voltammetry, and capillary
electrophoresis.[3-10] The disadvantages of these
techniques are the need for large quantities of
organic solvents, long analysis times and complex
sample preparation steps. Organic solvents such as
methanol, acetonitrile, trifluoroacetic acid, and
borate buffer are not only very expensive, but also
cause environmental damage. It is imperative to find
an analytical method that is not only fast and
accurate, but also does not cause environmental
pollution, in order to comply with the principles of
green chemistry.
Nowadays, vibrational spectroscopic techniques
are routinely being used for the authentication of
pharmaceutical products.[11-18] They have also been
applied extensively in the characterization,
identification and quantification of medicines.[19, 20]
Conventional Fourier transform infrared
spectroscopy (FTIR) is one of the most traditional
Vietnam Journal of Chemistry Nguyen Quoc Thang et al.
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 616
techniques for identifying pharmaceuticals.[16,17] The
use of FTIR requires skilled personnel because
preparing samples for a transmission measurement is
a rather complex task. The solid samples typically
must be pulverized, finely diluted with the IR-
inactive KBr and then pressed into KBr-pellets,
before analysis. The drawback of this approach is
that it is time-consuming, requires continuous drying
of KBr, and a hydraulic press to produce uniform
KBr pellets. The use of attenuated total reflection
(ATR) in combination with the FTIR technique is
recommended in order to overcome these
limitations. When ATR is used in conjunction
with FTIR, samples can be examined straightaway,
without further modification. All types of samples,
including solids, liquids, powders, pastes, tablets,
and fibers can be directly placed on the ATR crystals
for measurement. In addition, the ATR-FTIR
technique not only measures samples within a few
seconds, but also provides excellent data quality,
combined with high reproducibility. For these
reasons, ATR-FTIR has been widely used as a tool
in pharmacological research, pharmaceutical
analysis, as well as in other scientific fields in recent
years.[21-23]
The main objective of this study was to develop
a simple, rapid, cost-effective and environment-
friendly analytical method for the determination of
piroxicam in marketed tablet dosages for routine
quality control analysis, based on ATR-FTIR,
without using any solvents or toxic chemicals and
without any sample handling process.
2. MATERIALS AND METHODS
2.1. Material
Pharmaceutical grade piroxicam and placebo were
supplied from GLOMED Pharmaceutical Co., Ltd.
lactose monohydrate, Plasdone S-630, benzoic acid,
magnesium stearate and sodium starch glycolate
were purchased from Merck (Darmstadt, Germany).
2.2. Apparatus
All spectra were recorded on the Jasco FT/IR-4700
type A spectrophotometer (Japan) equipped with the
ATR platinum diamond sampling stage to provide
robustness and durability. The FTIR spectra were
recorded in the range between 4000 and 500 cm-1,
using 16 scans for each spectrum, with a resolution
of 4 cm-1. Spectral acquisition was done using the
Spectra Manager, Version 2.
2.3. Preparation of excipients
Excipients including lactose monohydrate, plasdone
S-630, benzoic acid, magnesium stearate, and
sodium starch glycolate were used. These excipients
are commonly used in piroxicam tablets. [24]
2.4. Validation of the FTIR method
The developed FTIR method was validated for
selectivity, linearity, precision and accuracy as
indicated in the International Council for
Harmonisation of Technical Requirements for
Pharmaceuticals for Human Use (ICH)
guidelines.[25,26]
2.4.1. Selectivity
For the selectivity studies, the excipients, pure
piroxicam, and pure piroxicam in excipients
(standard) were tested against potential
interferences, in order to investigate the specificity
of the method.
2.4.2. Calibration curve and linearity
The solid-state calibration curve was prepared by
mixing four different concentrations of pure
piroxicam (10, 20, 30, and 40 mg) with 387.2 mg of
excipients in about 10 minutes to reach a
homogeneous mixture, using a mortar and pestle for
homogenization. Before recording the FTIR
spectrum, each standard calibration powder was
diluted with potassium bromide powder. For each
calibration standard, three samples were prepared,
and each sample was analyzed in triplicate.
Recording the FTIR spectra, export to excel.xlsx
program, picked out the percent transmittance at
1526 cm-1. The percentage transmittance
corresponding to the secondary -NH peak centered
at 1526 cm-1 was plotted against the concentration of
piroxicam, and a linear regression analysis was
performed on the data to obtain the linear regression
equation. The resultant calibration curve was used to
quantify the amount of piroxicam present in the
tablet. The linearity of the calibration curve was
assessed by the coefficient of determination from the
linear regression analysis.
2.4.3. Precision
The precision of the method was evaluated by
repeatability and intermediate precision studies.
Repeatability (intra-assay) was established by
analyzing the marketed tablet sample (whose label
claimed to contain 20 mg of piroxicam) and
piroxicam standard (pure piroxicam added to
Vietnam Journal of Chemistry Attenuated total reflection-fourier transform
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 617
excipients) six times on the same day (day 1) and
under identical working conditions. The
intermediate precision studies (inter-assay) were
performed by repeating these studies inter-day (on
day 3), under the same experimental conditions. At
the end of the test, the precision was expressed by
the relative standard deviation percentage (%RSD).
2.4.4. Accuracy and recovery efficiency
Accuracy is a measure of the closeness of the
experimental value to the actual amount of the
piroxicam present in the excipients. The statistical
accuracy was determined by adding a known amount
(10.0, 15.0 or 20.0 mg) of pure piroxicam to the
excipients. The accuracy of the method was
evaluated by the recovery efficiency of pure
piroxicam from the excipients. The recovery
efficiency was assessed for three different added
concentrations, in six replicates.
The recovery efficiency (RE) (%) was determined
by the ratio of the piroxicam recovered (mg) to that
of the added piroxicam (mg). The following
equation represents the calculation:
RE(%) =
(C − B)
A
x 100%
where B (mg) is the actual concentration of
piroxicam present in the sample (before the addition
of a further amount of piroxicam), C (mg) is the
concentration of piroxicam after the addition and, A
(mg) is the concentration of the added piroxicam.
2.5. Analysis of marketed tablet dosage
Two different brands (each of whose labels claimed
to contain 20 mg of piroxicam) were used to
determine the piroxicam content. Twenty tablets of
each brand were weighed and ground into a fine
powder with a quartz pestle. About 0.01 g of tablet
powder was used for the analysis, carried out using
six samples which were evaluated in six replicates.
2.6. Justify the quantification method
Determination of piroxicam in two marketed tablets
using FTIR and reverse-phase high-performance
liquid chromatography with Diode Array Detector
(RP-HPLC/DAD) apparatus. Using the Statistical
Package for Social Sciences (SPSS) version 22.0 to
calculate the differences between means were
evaluated by Independent-Samples T Test and p <
0.05 was considered as significance.
3. RESULTS AND DISCUSSION
3.1. Quality control criteria for the FTIR
spectroscopy method
Using ATR-FTIR techniques in pharmaceutical
quality control offers significant benefits in terms of
fast, accurate, simple and green methods. Figure 2
represents the FTIR transmission spectrum of
piroxicam in the infrared region, as recorded by the
ATR mode.
Figure 2: Transmittance spectra of (a) pure piroxicam, (b) excipients (Lactose monohydrate, plasdone
S-630, benzoic acid, magnesium stearate, sodium starch glycolate) and (c) piroxicam in excipients
Vietnam Journal of Chemistry Nguyen Quoc Thang et al.
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 618
Figure 2a shows the FTIR spectrum for the pure
sample. The characteristic vibrational peaks at 3335,
1627 and 1572 cm-1, were assigned to the pyridin-2-
yl-amino, -NH-CO, and C=C stretching modes,
respectively. The peak at 1526 cm-1 was assigned to
the secondary -NH stretching mode. The bands at
1431 and 1347 cm-1 were attributed to the
asymmetrical C-H and the symmetrical C-H bending
modes, respectively. The characteristic vibrational
peaks at 1297, 1213 and 1145 cm-1, correspond to
the S=O symmetric, C-C and -SO2-N- stretching
modes respectively. The band at 768 cm-1 was
attributed to the ortho-disubstituted phenyl group.
The results obtained in this study were consistent
with literature.[27,28] The FTIR spectra of excipients
in Figure 2b shows that most vibrational peaks are
distinct from those of pure piroxicam. As can be
seen in the spectrum of piroxicam in the presence of
excipients (see Figure 2c), there are no significant
interference bands due to the excipients at the
wavenumbers corresponding to the characteristic
vibrational peaks of piroxicam, clearly indicating the
high degree of specificity and therefore, the
suitability of this approach for practical applications.
In the present study, the secondary -NH peak
centered at 1526 cm-1 was selected for quantification
of piroxicam in tablet dosages, due to the absence of
characteristic vibrational peaks attributed to
excipients in this region of the spectrum
Table 1: Linear regression data for piroxicam (n = 3)
Parameters Value
Slope -0.2178
Intercept 99.208
Linearity range 25.18-93.63 mg/g
Correlation
coefficient (r2)
0.9987
Standard error of
regression
3.18%
Thus, ATR-FTIR method is completely aligned with
the principles of green chemistry. That is, the
piroxicam content can be evaluated, without the use
of any solvent, whatsoever.
To assess the linearity of the calibration curve,
piroxicam was thoroughly mixed with powdered
excipients. The piroxicam concentration was then
analyzed in the range of 25.18–93.63 mg/g in
triplicates. The calibration curve in this range was
found to be linear, with the regression equation
given by y = -0.2178x + 99.208 and a correlation
coefficient (r2) of 0.9987 (see Figure 3 and Table 1).
This regression equation was later used to estimate
the concentration of piroxicam in the real samples.
Figure 3: (a) Transmittance spectra and (b) calibration curve for the piroxicam concentration in the
range of 25.18-93.63 mg/g
The precision of the method was expressed as
the percentage of the relative standard deviation
(%RSD). Repeatability was checked with multiple
measurements of the spectrum over three days of
two distinct types of piroxicam samples (standard
and tablet) at a concentration of 20 mg, with
subsequent calculation of the % RSD. These intra-
day and inter-day experiments were performed
under the same operating conditions, in the same
laboratory, for the same time period. As can be seen
in table 2, the % RSD values for the precision
studies are in the range of 2.53-3.15 % on day 1,
1.05-1.92 % on day 2 and 1.36-2.84 on day 3. Both
the intra-day and inter-day precision results were
within the accepted limits, in accordance with ICH
guidelines.
The most common way to evaluate the accuracy
of a proposed technique is the spike recovery
method. In the present study, a known quantity of
piroxicam (10.0, 15.0 or 20.0 mg) was spiked into
Vietnam Journal of Chemistry Attenuated total reflection-fourier transform
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 619
excipients. By comparing the amount of piroxicam
found and the amount of piroxicam spiked, the
recovery efficiency of the present method was
calculated. The statistics of piroxicam recovery
studies are compiled in table 3. The results indicate a
high recovery performance (96.0-99.3 %) with an
excellent %RSD (1.78-4.63 %) of the proposed
method, which are within acceptable limits as
specified by ICH guidelines. Furthermore, the
recovery percentage values obtained here indicate
that there is no significant interference effect from
any excipient present in the matrix. This also implies
that the method proposed here can be used to
estimate the content of piroxicam, without any need
for without solvent extraction.
Compare to the result of reversed-phase high-
performance liquid chromatographic (RP-HPLC)
method for the determination of piroxicam in tablets.
Table 2: Results for the evaluation of precision of piroxicam (n = 6)
Sample
Amount
added
(mg)
Day 1 Day 2 Day 3
Amount
found (mg)
% RSD
Amount found
(mg)
% RSD
Amount
found (mg)
% RSD
Standard 20.0 19.80±0.62 3.15 19.67±0.38 1.92 19.37±0.55 2.84
Tablet 20.0 19.87±0.50 2.53 19.73±0.21 1.05 19.50±0.26 1.36
Table 3: Recovery results of piroxicam from spiked samples (n = 5)
Excipient (mg) Added amount (mg) Found amount (mg) Recovery (%) % RSD
387.8 10.0 9.63±0.25 96.3 2.61
386.4 10.2 9.80±0.26 96.1 2.70
387.5 10.2 9.90±0.46 97.1 4.63
387.1 15.1 14.90±0.30 98.7 2.01
386.7 15.2 15.00±0.30 98.7 2.00
387.9 14.9 14.80±0.30 99.3 2.03
387.2 20.0 19.73±0.35 98.5 1.78
386.9 20.1 19.27±0.42 96.0 2.16
387.8 20.2 19.53±0.40 96.5 2.07
3.2. Quantification of piroxicam in marketed
tablet dosage
The ATR-FTIR method developed and validated in
this study was used to determine the quantity of
piroxicam present in two different marketed brands.
The results are summarized in table 4. The
persistence of the peak around 1526 cm-1 in the
FTIR spectrum of the marketed tablet dosages (see
figure 4) confirmed that there was no significant
interference due to the presence of excipients. The
average recoveries of piroxicam in the two marketed
tablet dosages are in the range of 95.6-97.4 % of
what was claimed on the labels, with %RSD values
in the range of 3.2-4.1 %. The recovery percentage
of piroxicam is within acceptable limits (95-105 %
in The British Pharmacopoeia Commission
Secretariat of the Medicines and Healthcare
Products Regulatory Agency).[29] This study
demonstrates that the simple ATR-FTIR approach
employed has the capacity to rapidly identify, as
well as estimate piroxicam content in the presence of
excipients. The whole process of crushing,
identifying and quantifying the piroxicam content of
a tablet would take about three minutes per tablet
sample.
Figure 4: FTIR spectrum of piroxicam in
marketed tablet dosage
The piroxicam level in two different marketed
brands were determined using HPLC and FTIR
method and the results of the independent-samples t-
Vietnam Journal of Chemistry Nguyen Quoc Thang et al.
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 620
test were shown in table 5. Two significance level
(2-tailed) in the table 5 were greater than 0.05, this
usually concluded that piroxicam level means in
HPLC and FTIR method were equal. The
concentration of piroxicam in two marketed tablets
were not significantly different between FTIR
method and HPLC method.
Table 4: The concentrations of piroxicam in marketed tablet dosage (n = 6)
Marketed tablet Label claim (mg) Observed (mg) % RSD Recovery (%)
Brand-1 20 19.12±0.14 3.2 95.6
Brand-2 20 19.49±0.12 4.1 97.4
Table 5: The independent-samples t-test compares the means of piroxicam level
From HPLC and
FTIR results
Marketed tablet
t-test for equality of means
HPLC method
(mg)
FTIR method
(mg)
t value
Significance level
(2-tailed)
Brand-1 19.20±0.11 19.12±0.14 1.019 0.338
Brand-2 19.31±0.13 19.49±0.12 2.282 0.052
The method developed and validated in the
present study emphasizes the accuracy and
effectiveness of using ATR-FTIR spectroscopy to
identify and analyze drugs. The most outstanding
advantages of this method are enumerated below:
(i) it is a fast analysis technique,
(ii) sample preparation is not necessary,
(iii) conforms to green chemistry standards,
since no solvent or toxic chemical need be used, and
(iv) has a remarkably high recovery efficiency
(96.0 % to 99.3 %).
4. CONCLUSIONS
The ATR-FTIR is a useful, economical and eco-
friendly method to estimate piroxicam content in
tablet dosage forms since it is simple, rapid, precise,
and accurate, w