DIFFERENTIAL SCANNING CALORIMETRY AND INFRARED SPECTROMETRY METHODS FOR THE QUANTIFICATION OF ETHAMBUTOL HYDROCHLORIDE IN ORAL DOSAGE FORMS
HTML Full TextDIFFERENTIAL SCANNING CALORIMETRY AND INFRARED SPECTROMETRY METHODS FOR THE QUANTIFICATION OF ETHAMBUTOL HYDROCHLORIDE IN ORAL DOSAGE FORMS
N. Delhiraj * and A. M. Sekar
Department of Pharmaceutical Analysis, School of Pharmacy, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India.
ABSTRACT: A new, simple, and sensitive differential scanning calorimetry and infrared spectrophotometric technique was developed to estimate ethambutol hydrochloride in tablet dosage form. In the first method the concentration range of ethambutol hydrochloride was discovered to be 0.5 mg to 2.0 mg using differential scanning calorimetry (DSC), with the peak region of an exothermic thermogram at 75.8 °C and a continuous heating rate of 5 °K min-1. Second method which involves the measurement of ethambutol hydrochloride by Infra Red (IR) spectroscopy, the concentration range was determined to be 1.0 mg to 2.5 mg. The correlation coefficients for these techniques were determined to be 0.999 and 0.9919 for both DSC and IR methods respectively. The analytical results were statistically validated by the ANOVA technique and recovery trials. The recovery trials show no influence from other chemicals in the formulation. As a result, these two procedures are easy, precise, accurate and specific, and they might be employed for regular analysis.
Keywords: Beer's law concentration, Ethambutol hydrochloride, Differential scanning Calorimetry (DSC), Infra red spectroscopy (IR).
INTRODUCTION: Ethambutol hydrochloride 1 is a chemotherapeutic agent that has been found to be effective against Mycobacterium tuberculosis Fig. 1. It is used as the first line of defence against pulmonary tuberculosis, along with other anti-tubercular drugs such as rifampicin, isoniazid, and pyrazinamide. Ethambutol was developed in response to a need for antibiotics that were effective against isoniazid-resistant Mycobacterium tuberculosis strains.
Ethambutol enters cells and inhibits arabinosyltransferases (embA, embB, and embC), preventing the formation of cell wall components such as arabinogalactan and lipoarabinomannan, resulting in cell division inhibition, mycolic acid accumulation, trehalose monomycolate and trehalose dimycolate accumulation, and interference with mycobacterial interaction with host cells.
According to the published literature 2-16 to determine the drug in raw materials, dosage forms, and biological fluids, a few sophisticated analytical techniques such as High-Performance Liquid Chromatography, UV spectrophotometry, and electrophoretic methods were used. Most of these methods include more time-consuming and labor-intensive phases, such as extraction and derivatization. As a result, the current study describes a novel, simple, and precise Fourier Transform- Infrared spectrophotometric method 17, 18. Differential Scanning Calorimetry method 19, 20 for detecting the content in bulk and pharmaceutical dosage formulations.
FIG. 1: STRUCTURE OF ETHAMBUTOL
MATERIALS AND METHODS:
Chemical and Reagents: Ethambutol reference standard obtained from by Orchid Pharma in Chennai, India. IR grade potassium bromide (KBr) and Analytical grade internal standard potassium thiocyanate (KSCN) were given by Merck (Darmstadt, Germany).
Method A: Differential Scanning Calorimetry Method:
Preparation of Standard and Sample Solutions: To determine the content, the calibration curve was plotted. Four distinct dosages of standard drug (0.5mg-2.0mg) were properly weighed for the DSC experiment. The experiment was conducted in a stream of nitrogen (about 50 cm3 min-1) at a heating rate of 5 K min-1 in a temperature range of -4°C to 174°C using the Netzsch DSC-204 and Proteus software, with an empty aluminum pan serving as a reference. The calibration curve was generated by plotting the peak area at 75.80°C (exothermic thermogram) versus concentration.
Preparation of Standard and Sample Solutions: 20 tablets were weighed and crushed to a fine powder, Tablet powder was accurately weighed equivalent to 1.0 mg of ethambutol and used for DSC thermogram recording. Interpolating the sample peak area on the calibration curve at 75.80°C (exothermic thermogram) yielded drug concentration in the formulation.
Method B: IR-KBr Disc Method:
Preparation of Standard Solutions: Potassium thiocyanate (KCNS) was preground, dried, then reground with dry KBr to obtain a thiocyanate concentration of roughly 0.2% by weight as an internal standard. The final mixture was kept in the presence of phosphorous pentoxide.
For the calibration curve, known weights of the standard substance were mixed with a known weight of the KBr-KCNS mixture, as shown in Table 1, pipetting out the required quantity in a china dish from a 20% w/v alcoholic solution of the standard drug, and evaporating the solvent from the residue. A known amount of the KBr-KCNS mixture was added, dried using an infrared light, and homogenised with an agate mortar and pestle.
The discs were manufactured using a KBr press and a hydraulic press (Model no. CAP-15T), and the infrared spectrum was captured using an Agilent in absorbance mode. Technology instrument (FTIR model Cary 630). The calibration curve was generated by plotting the absorbance of the IR absorption at 2971 cm-1 (the dominant band) against the chemical concentration.
TABLE 1: CONCENTRATION OF KBR/KCNS MIXTURE AND STANDARD
KBr/KCNS (mg) | 50 | 50 | 50 | 50 | 50 |
Reference Standard (mg ) | 0.0 | 1.0 | 1.5 | 2.0 | 2.5 |
Preparation of Sample Solutions: 20 tablets were weighed, the average weight was established, and the pills were ground to a fine powder. A tablet powder containing 1.5mg of drug was precisely weighed and mixed with the KBr-KCNS mixture before being homogenised with a mortar and pestle. The resultant powder was transferred to a KBr press with a Hydraulic press (Model no.CAP-15T) to form a disc, and the infrared spectrum was recorded in absorbance mode with an Agilent Technology Instrument (Model no. Cary 630 FTIR). The concentration was estimated by interpolating the sample absorbance using the EMB's linearity curve.
Recovery Studies: The recovery studies were carried out using spiked samples, which were created by adding a predetermined amount of standard medicines to the respective sample. After 50 and 100% of standard drugs were supplied, the absorbance and peak area of the sample were measured. The recovery percentage was calculated. The recovery study was undertaken at two levels to check the precision and correctness of the above-mentioned procedures.
RESULTS AND DISCUSSION: In DSC analysis, Fig. 2 depicts the DSC thermogram of various concentrations of reference standard and oral dosage form at a heating rate of 5 k min-1 and chilling under a nitrogen stream. Fig. 3 shows the calculated peak area value for the reference standard, as well as the calibration curve between the peak area at 75.80 degrees Celsius (Exothermic thermogram) and concentration. The correlation coefficient for EMB was calculated to be 0.9993 using equation line y= 47.668x+1.81. The linearity values of peak area at 75.8℃ (Exothermic thermogram) versus concentration are shown in Table 2.
FIG. 2: DSC THERMOGRAM OF DIFFERENT CONCENTRATION OF ETHAMBUTOL HYDROCHLORIDE REFERENCE STANDARD AND ORAL DOSAGE FORM
TABLE 2: THE AREA OF PEAK AT 75.8℃ EXOTHERMIC THERMOGRAM
S. no. | Concentration of Drug | Peak Area* |
1 | 0.5 mg | 25.46 |
2 | 1.0 mg | 50.12 |
3 | 1.5 mg | 72.58 |
4 | 2.0 mg | 97.42 |
*Each value is the mean of three determinations.
FIG. 3: CALIBRATION CURVE FOR STANDARD ETHAMBUTOL
In IR analysis, Fig. 4 illustrates the IR spectra of drug in conventional and oral dosage forms. Fig. 5 represents the calibration curve drawn between concentration and absorbance value, while Table 3 gives the absorbance value of standard at wavelength 2971 cm-1. With the equation line y= 538x+0.036, the coefficient of correlation was calculated to be 0.9919.
FIG. 4: IR SPECTRA OF STANDARD & ORAL DOSAGE FORM ETHAMBUTOL HYDROCHLORIDE
TABLE 3: CONCENTRATION OF A PEAK AREA AT 2971 CM-1 WAVE NUMBER
S. no. | Concentration of Drug | Absorbance |
1 | 1.0 mg | 0.62 |
2 | 1.5 mg | 0.77 |
3 | 2.0 mg | 1.12 |
4 | 2.5 mg | 1.40 |
*Each value is the mean of three determinations.
FIG. 5: CALIBRATION CURVE OF ETHAMBUTOL VERSUS PEAK AREA
Table 4 shows the optical properties, such as the concentration range of Beer's law. Table 4 summarizes the findings of the regression features such as slope (b), intercept (c), and correlation coefficient. The recovery percentages of the two procedures range between 98 and 100% w/w. The correlation values for the two procedures were 0.999 and 0.9919, respectively, and the recovery trials show no influence from other chemicals in the formulation. As a result, these two procedures are easy, precise, accurate, time-consuming, and specific, and they might be employed for regular analysis. Table 5 shows the test results obtained using the proposed method. Validation studies for the suggested methodologies were conducted, and the results are shown in Tables 6 and 7. DSC and IR measurements do not require any prior extraction and are unaffected by drug solubility.
TABLE 4: OPTICAL PARAMETERS OF ETHAMBUTOL HYDROCHLORIDE BY DSC AND IR METHOD
Parameters | Method A
Heat-Flux DSC method |
Method B
KBr Disc method using Internal standard |
Beer’s law limit (mg) | 0.5– 2.0 | 1.0 – 2.5 |
Regression equation (y = mx + c ) | 47.668x+1.81 | 0.538x+0.036 |
Slope (m) | 47.668 | 0.538 |
Intercept (C ) | 1.81 | 0.036 |
Correlation coefficient | 0.9993 | 0.9919 |
LOD (µg/mL) | 0.250608 | 0.441636 |
LOQ (µg/mL) | 0.759419 | 1.33829 |
TABLE 5: RESULT OF TABLET ASSAY
S. no. | Method | Label claim | Amount found (mg)* | Percentage Assay | SD | SE | %RSD |
1 | Method A | 100 mg | 99.02 | 99.02 | 0.541223 | 0.31249 | 0.546553 |
2 | Method B | 100 mg | 99.56 | 99.56 | 1 .495337 | 0.863359 | 1.501895 |
*Each value is a mean of 3 determinations.
TABLE 6: RECOVERY STUDY
S. no. | Method | Label claim | Amount of drug added (mg)* | Amount of drug recovered (mg)* | Percentage Recovery |
1 | Method A | 100mg | 0.5
1.0 |
0.496
0.994 |
99.20
99.46 |
2 | Method B | 100mg | 0.75
1.5 |
0.742
1.48 |
98.93
98.66 |
*Each value is a mean of 3 determinations.
TABLE 7: ANOVA CALCULATION FOR DSC METHOD
Source of Variation | SS | Df | MS | F-Ratio | P-value* |
Between sample | 7234.8421 | 1 | 7234.8421 | 15.27087 | 0.007912 |
Within sample | 2842.6047 | 6 | 473.7675 |
*The result is significant of P < 0.05.
TABLE 8: ANOVA CALCULATION FOR IR METHOD
Source of Variation | SS | Df | MS | F-Ratio | P-value* |
Between sample | 1.5051 | 1 | 1.5051 | 6.41896 | 0.044465 |
Within sample | 1.4069 | 6 | 0.2345 |
*The result is significant of P < 0.05.
CONCLUSION: The proposed methods for measuring the drug, ethambutol hydrochloride in bulk and pharmaceutical dose forms are simple, precise, exact, time-efficient, specific, and selective.
In contrast to the chromatographic technique, these methods are less expensive and faster, and they do not need complex equipment. As a result, it is suitable for regular analysis in bulk and medicinal dose forms.
ACKNOWLEDGEMENT: The authors are thankful to the Sophisticated Analytical Instrument Facility- Indian Institute of Technology (SAIF-IIT) Chennai for extending the laboratory facilities and supporting the research work.
CONFLICT OF INTEREST: The authors of this research work have no conflict of interest
REFERENCES:
- Zhu C, Liu Y, Hu L, Yang M and He ZG: Molecular mechanism of the synergistic activity of ethambutol and isoniazid against Mycobacterium tuberculosis. J Biol Chem 2018; 293(43): 16741-16750.
- Abouzid M, Kosicka-Noworzyń K, Karaźniewicz-Łada M, Rao P, Modi N, Xie YL, Heysell SK, Główka A and Kagan L: Development and validation of a uplc-ms/ms method for therapeutic drug monitoring, pharmacokinetic and stability studies of first-line antituberculosis drugs in urine. Molecules 2024; 29(2): 337. doi: 10.3390/ molecules 29020337.
- Oliveira MAL, Chellini PR and Amorim TL: Simultaneous determination of rifampicin, isoniazid, pyrazinamide and ethambutol in fixed dose combination antituberculosis pharmaceutical formulations: a review: Analytical Methods 2017; 00: 1-7 DOI: 10.1039/C7AY02686B
- Panda BK and Bargaje MLS: A simple and reliable analytical method for simultaneous quantification of first line antitubercular drugs in human plasma by LCMS/MS. Anal Methods 2020; 12(31): 3909-3917. doi: 10.1039/d0ay00889c.
- Zheng X, Jongedijk EM, Hu Y, Kuhlin J, Zheng R, Niward K, Paues J, Xu B, Davies Forsman L, Schön T, Bruchfeld J and Alffenaar JC: Development and validation of a simple LC-MS/MS method for simultaneous determination of moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2020; 1158: 122397. doi: 10.1016/j.jchromb.2020.122397.
- Zheng Y, Xu N, Hu X, Zhang Q, Liu Y and Zhao Q: Development and Application of a LC-MS/MS Method for Simultaneous quantification of four first-line antituberculosis drugs in human serum. J Anal Methods Chem 2020; 2020: 8838219. doi: 10.1155/2020/8838219.
- Anjani QK, Bin Sabri AH and Donnelly RF: Development and validation of simple and sensitive HPLC-UV method for ethambutol hydrochloride detection following transdermal application. Anal Methods 2022; 14(2): 125-134. doi: 10.1039/ d1ay01414e.
- Sri Lakshmi D and Jane T Jacob: Validated degradation studies for the estimation of Pyrazinamide, Ethambutol, Isoniazid and Rifampacin in a fixed dose combination by UPLC. Research J. Pharm. and Tech 2018; 11(7): 2869-2875. doi: 10.5958/0974-360X.2018.00529.2
- Shewiyo DH, Kaaleb E, Rishab PG, Dejaegherc B, Smeyers-Verbekec J and Heydenc YV: Optimization of a reversed phase high performance thin layer chromatography method for the separation of isoniazid, ethambutol, rifampicin and pyrazinamide in fixed-dose combination antituberculosis tablets. J Chromatogr A 2012; 1260: 232-8. doi: 10.1016/j.chroma.2012.08.044.
- Chellini PR, Lages EB, Franco PHC, Nogueira FHA, César IC and Pianetti GA: Development and Validation of an HPLC Method for Simultaneous Determination of Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol Hydrochloride in Pharmaceutical Formulations. Journal of AOAC International 2015; 98: 1234-1239. doi:10.5740/jaoacint.14-237
- Hagga MAM and Sultana S: A Novel Quantitative Method for the Simultaneous Assay of Rifampicin (RIF), Isoniazid (INH), Ethambutol (EMB), and Pyrazinamide (PYP) in 4-FDC Tablets. Oriental Journal of Chemistry 2016; 32: 3081-3087. doi:10.13005/ojc/320629.
- Franco PHC, Chellini PR, Oliveira MAL and Pianetti GA: Simultaneous Determination of First-Line 4-FDC Antituberculosis Drugs by UHPLC–UV and HPLC–UV: A Comparative Study, Journal of AOAC International 2017; 100(4): 1008–1015. doi :10.5740/jaoacint.16-0200
- Marcellos LF, Faria AF, Souza MVN, Almeida MR, Sabin GP, Poppi RJ and Oliveira MAL: Simultaneous analysis of first-line anti-tuberculosis drugs in tablets by UV spectrophotometry compared to capillary zone electrophoresis. Central European Journal of Chemistry 2012; 10: 1808-1816.doi:10.2478/s11532-012-0102-6
- Faria AF, Souza MVN, Bruns RE and Oliveira MAL: Simultaneous determination of first-line anti-tuberculosis drugs by capillary zone electrophoresis using direct UV detection, Talanta 2010; 82(1): 333-339 doi.org/10.1016/j.talanta.2010.04.044.
- Neves ACDO, Soares GM, Morais SCD, Costa FSLD, Porto D and Lima KMGD: Dissolution testing of isoniazid, rifampicin, pyrazinamide and ethambutol tablets using near-infrared spectroscopy (NIRS) and multivariate calibration. Journal of Pharmaceutical and Biomedical Analysis 2012; 57: 115-119. doi:10.1016/j.jpba. 2011.08.029
- Moreno ADH and Salgado HRN: Development and validation of the quantitative analysis of ceftazidime in powder for injection by infrared spectroscopy, Physical Chemistry 2012; 2(1): 6-11.
- Fatmarahmi DC, Susidarti RA, Swasono RT and Rohman A: Identification and quantification of metamizole in traditional herbal medicines using spectroscopy ftir-atr combined with chemometrics. Research Journal of Pharmacy and Technology 2021; 14(8): 4413-9. doi: 10.52711/0974-360X.2021.00766
- Teixeira KSS, Fonseca SGC, Moura LCB, Moura MLR, Borges MHP, Barbosa EG and Moura TFAL: Use of chemometrics to compare NIR and HPLC for the simultaneous determination of drug levels in fixed-dose combination tablets employed in tuberculosis treatment. Journal of Pharmaceutical and Biomedical Analysis 2018; 149: 557–563
- Gumieniczek A, Berecka-Rycerz A, Trębacz H, Barzycka A, Leyk E and Wesolowski M: DSC, FT-IR and NIR with chemometric assessment using PCA and HCA for estimation of the chemical stability of oral antidiabetic drug linagliptin in the presence of pharmaceutical excipients. Molecules 2022; 27(13): 4283. doi: 10.3390/ molecules 27134283.
- Kodre KV, Attarde SR, Yendhe PR, Patil RY and Barge VU: Differential scanning calorimetry: a review. Research and Reviews J of Pharma Analysis 2014; 3(3): 11-22.
How to cite this article:
Delhiraj N and Sekar AM: Differential scanning calorimetry and infrared spectrometry methods for the quantification of ethambutol hydrochloride in oral dosage forms. Int J Pharm Sci & Res 2024; 15(7): 2058-63. doi: 10.13040/IJPSR.0975-8232.15(7).2058-63.
All © 2024 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
19
2058-2063
600 KB
150
English
IJPSR
N. Delhiraj * and A. M. Sekar
Department of Pharmaceutical Analysis, School of Pharmacy, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India.
pharmaraj1981@gmail.com
21 January 2024
15 March 2024
16 April 2024
10.13040/IJPSR.0975-8232.15(7).2058-63
01 July 2024