Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) for quantification of piroxicam in tablet dosage from as a green analytical method

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