Isoniazid is an antitubercular drug, widely used for tuberculosis. Owing to its importance in therapeutics, the present study aims to develop simple method for the spectrophotometric determination of isoniazid (INH). Two novel reagents, epichlorohydrine (ECH) and 4-hydroxyphenaylchloride (HPC) are used for the spectrophotometric determination of INH. Based on the nucleophilic substitution reactions of INH with EPI & HPC in basic medium, rapid, simple, inexpensive, precise, and accurate visible spectrophotometric method is proposed for the determination of INH in bulk drug and in formulations. Method involves the reaction of INH with EPI and HPC in basic medium to form yellow-colored chromogen, measuring the absorbances at 405 and 402 nm for INH-EPI & INH-HPC, respectively. The optimum experimental conditions have been studied. The absorbance was found to increase linearly with the concentration of the drug and formed the basis for quantification. The calibration graphs were linear from 2.00–22.00
The enhanced prevalence of infectious diseases threatens world population. Tuberculosis (TB) is characterized as a chronic bacterial infection caused by a germ called
Among the many drugs discovered for the treatment of TB, isoniazid (INH) is one of the powerful drug candidates. The discovery of INH was based on the nicotinamide activity against tubercle bacilli in the animal model observed by Chorine in 1945 [
INH is still designated as an essential antituberculosis agent by the World Health Organization (WHO), and is now largely used together with rifampicin and streptomycin for the chemotherapy of TB. This has prompted many investigators to plan methods for the rapid determination of INH in its pure form as well as in pharmaceutical preparations. There are various analytical procedures for the assay of INH, the most important being titrimetry [
A UV-Visible spectrophotometer (SHIMADZU, Model no.: UV-2550) with 1 cm matched quartz cells was used for the absorbance measurements.
All the reagents used were of analytical reagent grade. The solutions of EPI in ethanol (10%), HPC in ethanol (0.2%), and NaOH (1 M) were prepared. A 1000
Aliquots containing 2.00–22.00
Aliquots containing 20.00–120.00
The proposed method has been applied successfully for the determination of INH in some pharmaceutical formulations. Commercial INH tablets (Solonex and Isokin) were analyzed using the developed method. To minimize a possible variation in the composition of the tablets, the mixed contents of 20 tablets were weighed and ground, then the powder equivalent to 300 mg INH was dissolved in water by stirring for 10 min and filtered through Whatman No. 41 filter paper. Solutions of working concentration were prepared by proper dilution of this stock solution with water and followed the above procedures for the analysis.
The proposed method is based on the nucleophilic substitution reaction of EPI in presence of NaOH to form yellow-colored chromogen (Scheme
Reaction of INH with ECH.
Absorption spectrum of INH-EPI system.
Preliminary experiments are carried out to fix the initial concentration of the reagents. The influence of the volume of 0.25 M NaOH on the formation of yellow color is studied. This is performed by keeping other parameters constant and taking different volumes of (0.1–5.0 mL) of 0.25 M NaOH. The maximum absorbance is obtained with 1 mL of NaOH. Above this volume absorbance remains constant. Therefore, this volume is used for all the absorbance measurements. To investigate the optimum heating time for color development, the content of the mixture is heated on water bath at 60°C for 5–10 min. The maximum intensity of color is obtained at 5 min of heating at 60°C and remains constant. To study the effect of concentration of EPI, different volumes of 10% ECH are tested. It is found that 1 mL of EPI is sufficient for very good color intensity.
The method is based on the nucleophilic substitution reaction of HPC with isoniazid in presence of NaOH (Scheme
Reaction of INH with HPC.
Absorption spectrum of INH-HPC system.
Preliminary experiments were carried out to fix the initial concentration of the reagents. The influence of the volume of 1 M NaOH on the formation of yellow color is studied. This is performed by keeping other parameters constant and different volumes of (0.1–5.0 mL) of 1 M NaOH. The maximum absorbance is obtained with 1 mL of NaOH. Above this volume absorbance remains constant. Therefore this volume is used for all the absorbance measurements. To investigate the optimum heating time for color development, the content of the mixture is heated on a water bath at 60°C for 5–30 min. The maximum intensity of color is obtained at 15 min of heating and remains constant. To study the effect of concentration of HPC, different volumes of 0.2% HPC are tested. It is found that 1 mL of HPC is sufficient for very good color intensity.
The linearity between two parameters is apparent from the correlation coefficient obtained by the method of least squares. The optical characteristics such as absorption
Analytical parameters.
EPI | HPC | |
---|---|---|
|
405 | 402 |
Beer’s law limit ( |
2.00–22.00 | 20.00–120.00 |
Molar absorptivity (L mol−1 cm−1) | 0.51 × 104 | 0.10 × 104 |
Sandell’s sensitivity ( |
0.027 | 0.134 |
Regression equation* |
|
|
Slope ( |
0.0336 | 0.0064 |
Intercept ( |
0.022 | 0.018 |
Correlation coefficient ( |
0.9910 | 0.9979 |
Limit of Detection** ( |
1.500 | 5.150 |
Limit of Quantitation** ( |
4.545 | 15.620 |
*
** Calculated using ICH guidelines.
Validation of an analytical procedure is the process by which it is ascertained, by laboratory studies, that the performance characteristics of the procedure meet the conditions for its proposed use. All analytical methods planned to be used for analyzing any experimental samples will need to be authenticated. The accuracy of the method was established by analyzing the pure drugs at diverse levels within working limits and the precision is ascertained by calculating the relative standard deviation of replicate determinations on the same solution containing the drugs at different levels and are presented in Tables
(a) Evaluation of accuracy and precision (using ECH as reagent). (b) Evaluation of accuracy and precision (using HPC as reagent).
Amount taken ( |
Amount found* ( |
RE (%) | SD ( |
RSD (%) |
---|---|---|---|---|
2.00 | 1.98 | 1.00 | 0.015 | 0.79 |
4.00 | 3.97 | 0.75 | 0.0207 | 0.52 |
6.00 | 5.96 | 0.66 | 0.0239 | 0.4 |
8.00 | 7.97 | 0.37 | 0.0259 | 0.32 |
10.00 | 9.96 | 0.4 | 0.0270 | 0.27 |
12.00 | 11.95 | 0.41 | 0.0404 | 0.33 |
* Mean of five determinations.
Amount taken ( |
Amount found* ( |
RE (%) | SD ( |
RSD (%) |
---|---|---|---|---|
20.00 | 19.99 | 0.05 | 0.09 | 0.45 |
40.00 | 40.03 | −0.08 | 0.06 | 0.14 |
60.00 | 59.96 | 0.06 | 0.12 | 0.20 |
80.00 | 79.93 | 0.08 | 0.08 | 0.10 |
* Mean of five determinations.
RE: relative error; SD: standard deviation; RSD: relative standard deviation.
The specificity of an analytical method may be defined as the ability to clearly determine the analyte in the presence of additional components such as impurities, degradation products, and matrix. The specificity in the current case is evaluated by preparing the analytical placebo and it is confirmed that the change in absorbance with respect to the reagent blank is caused only by the analyte. A solution of the analytical placebo (containing all the tablet excipients except INH) is prepared according to the sample preparation procedure and subjected to analysis using the procedures described earlier. The absorbance measured is nearly the same as that of the reagent blank. To identify the interference by these excipients, a synthetic mixture of inactive ingredients (placebo) including INH with the following composition: INH (10 mg), talc (20 mg), starch (40 mg), glucose (50 mg), and lactose (40 mg) is prepared. The entire mixture is transferred into a 100 mL calibrated flask, the content shaken for 20 min, volume diluted to the mark with distilled water, mixed well, and filtered. The filtrate after suitable dilution is analyzed by proposed methods. The difference between the measured absorbance of the above extract and that of a standard INH solution of the same concentration is less than 2% indicating the absence of interference by the excipients.
The proposed method has been applied to the determination of INH in pure and dosage form. The results are compared statistically with those of the tabulated value at 95% confidence level. The calculated student’s
Results of assay of formulations.
Brand name | aIsokin | bSolonex |
---|---|---|
Labeled amount (mg) | 300 | 300 |
| ||
(1) Using ECH | ||
(i) Amount found* (mg) | 299.5 | 299.0 |
(ii) % Label claim ± SD | 99.83 ± 0.010 | 99.66 ± 0.018 |
(iii) |
2.23 | 2.48 |
| ||
(2) Using HPC | ||
(i) Amount found* (mg) | 299.70 | 299.81 |
(ii) % Label claim ± SD | 99.90 ± 0.08 | 99.93 ± 0.06 |
(iii) |
1.67 | 1.62 |
*Mean value of five determinations.
aPfizer Ltd., India.
bMacleods Pharmaceuticals Ltd.
Calculated Student’s
The new approach of utilizing epichlorohydrine and 4-hydroxyphenayl chloride as reagents in spectrophotometry is the first of such reports. The method makes use of very easily available and cheaper reagents which demonstrates its cost-effectiveness. Compared to many existing instrumental methods for INH, the proposed spectrophotometric method has two additional advantages of simplicity of operations and low-cost instrument. These advantageous features advocate its use in quality control laboratories for routine use.
B. Narayana thanks the UGC for financial assistance through SAP and BSR one-time grant for the purchase of chemicals. Divya N. Shetty thanks the UGC-RFSMS scheme (under SAP-Phase1) for providing financial help for the research work.