Two spectrophotometric methods are described for the determination of isoniazid (INH) in pharmaceuticals. In the first method (FCR method), INH is reacted with Folin-Ciocalteu reagent in Na2CO3 medium and the resulting blue colored chromogen measured at 760 nm. Iron(II), formed as a result of reaction between INH and iron(III), is made to react with ferricyanide, and the resulting Prussian blue is measured at 760 nm, basing the second method (FFC method). The conditions for better performance are optimized. Beer’s law is obeyed in the concentration ranges 0.5–10 and 0.2–3.0
Isoniazid (INH) (Figure
Structure of INH.
The drug is official in Indian Pharmacopoeia (IP) [
Visible spectrophotometry is by far the most widely used technique for the assay of INH. Methods were based on a variety of reaction schemes such as nucleophilic substitution [
The aim of this work was to investigate the utility of F-C reagent and iron(III) and ferricyanide systems in the assay of INH. The methods had sufficiently good accuracy and precision and presented a simple and time-saving assay of INH.
A Systronics model 166 digital spectrophotometer (Ahmedabad, India) with matched 1-cm quartz cells was used for absorbance measurements.
Chemicals used were of analytical reagent grade. Distilled water was used throughout the investigation. Pharmaceutical grade INH certified to be 99.85% pure was kindly provided by Cipla India Ltd., Bangalore, India, and was used as received. Isokin-300 (Pfizer Ltd., Gandhinagar, Hyderabad, India) tablets containing isoniazid 300 mg with vitamin B6 10 mg were purchased from local commercial store.
Aqueous solution of Folin-Ciocalteu reagent (1 : 1 v/v) was prepared by mixing 50 mL of reagent (Merck, Mumbai, India) with 50 mL water.
A 20% solution of sodium carbonate was prepared by dissolving 20 g compound (S.D. Fine Chem. Ltd., Mumbai, India) in 100 mL water.
A 500
A solution of 0.5 M FeCl3·6H2O was prepared by dissolving 14 g of the chemical (S.D. Fine Chem. Ltd., Mumbai, India) in 100 mL of 0.1 M HCl (Merck, Mumbai, India).
A stock standard solution of INH (100
Into a series of 10 mL volumetric flasks, different aliquots of working standard INH solution (20
Different aliquots of standard INH solution (10
Standard graph was prepared by plotting the absorbance
Twenty tablets were weighed and ground into a fine powder. An accurately weighed quantity containing 10 mg of INH was transferred to a 100 mL volumetric flask, 60 mL water added, shaken well for 20 minutes and made up to mark with water, and then filtered. This solution was diluted to a 20 and 10
A placebo blank containing starch (10 mg), acacia (15 mg), hydroxyl cellulose (10 mg), sodium citrate (10 mg), talc (20 mg), magnesium stearate (15 mg), and sodium alginate (10 mg) was prepared by mixing all components into a homogeneous mixture. 10 mg of the placebo blank was accurately weighed and its solution was prepared as described under Procedure for Pharmaceutical Tablets and then subjected to analysis by following the general procedure.
An accurately weighed 10 mg of INH was added to 10 mg of placebo blank and homogenized. Synthetic mixture was quantitatively transferred into a 100 mL volumetric flask and the extract was prepared as described under the Procedure for Pharmaceutical Tablets. The resulting extract was diluted to get 20 and 10
The method was validated according to the procedures described in ICH guidelines [
The intraday precision was evaluated by analyzing INH solution at three different levels. Similarly the interday precision was evaluated on five consecutive days (
Method robustness was studied by making small changes in the optimized experimental variables and their effect on the absorbance was evaluated by calculating the percentage RSD values. In order to determine the method ruggedness, analyses were performed using three instruments and also by three analysts with the same instrument.
The proposed FCR method is based on the formation of a blue colored chromogen, following the reduction of phosphomolybdotungstic mixed acid of the F-C reagent [
INH probably affects reduction of oxygen atoms from tungstate and/or molybdate in the F-C reagent by producing one or more possible reduced species which have characteristic intense blue color.
The FFC method involves the redox reaction of INH with ferric chloride, in the presence of potassium ferricyanide, under mild acidic conditions, to produce a blue colour with maximum absorption at 760 nm. The first step in the colour development is the reduction of iron(III) of ferric chloride to iron(II) which subsequently reacts with ferricyanide to form Prussian blue.
The intensely blue colored product (molybdenum-tungsten mixed acid blue in FCR method and Prussian blue in FFC method) formed in both methods exhibited maximum absorption at 760 nm. The absorption spectra of the blue colored products and of the reagent blanks are shown in Figures
(a) Absorption spectra of A: reaction product of INH (6
A series of preliminary experiments necessary for rapid and quantitative formation of colored products to achieve the maximum stability and sensitivity were performed. Optimum condition was achieved by varying one parameter at a time while keeping other parameters constant and observing its effect on the absorbance at 760 nm.
Several experiments were carried out to study the influence of F-C reagent concentration on the color development and the obtained results are shown in Figure
(a) Effect of volumes of F-C reagent (1 : 1) on the reaction product with INH (6
To select a suitable medium for the reaction, different aqueous bases such as sodium hydroxide, sodium carbonate or bicarbonate, sodium acetate, and sodium hydrogen phosphate were investigated. Better results were obtained with sodium carbonate. In order to determine the optimum concentration of Na2CO3, different volumes of 20% Na2CO3 solution (0–5 mL) were attempted at a constant concentration of INH (6
Maximum color development was obtained in 10 min after mixing the reactants, and the color was stable for at least 60 min thereafter (Figure
To optimize the concentrations of ferricyanide and ferric chloride reagents, different volumes of these reagents were used with a fixed concentration of INH. Constant absorbance was found with 1.0 mL of 0.5 M FeCl3 and 1.5 mL of 500
(a) Effect of volume of FeCl3 for method B (1.5
Colour development was complete in 10 min and stable for the next 90 min (Figure
A linear correlation was found between absorbance at
Sensitivity and regression parameters.
Parameter | Method A | Method B |
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760 | 760 |
Color stability, min | 60 | 90 |
Linear range, |
0.5–10.0 | 0.2–3.0 |
Molar absorptivity ( |
1.12 × 104 | 4.55 × 104 |
Sandell sensitivity*, |
0.0123 | 0.003 |
Limit of detection (LOD), |
0.03 | 0.14 |
Limit of quantification (LOQ), |
0.09 | 0.42 |
Regression equation, |
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Intercept ( |
0.0131 | 0.0198 |
Slope ( |
0.0905 | 0.3176 |
Standard deviation of |
9.98 × 10−2 | 9.98 × 10−2 |
Standard deviation of |
1.24 × 10−2 | 3.98 × 10−2 |
Regression coefficient ( |
0.9971 | 0.9979 |
The precision and accuracy of the proposed method were studied by repeating the experiment seven times within the day to determine the repeatability (intraday precision) and five times on different days to determine the intermediate precision (interday precision). The assay was performed for three levels of analyte in this method. The results of this study are summarized in Table
Evaluation of intraday and interday accuracy and precision.
Method | INH |
Intraday accuracy and precision ( |
Interday accuracy and precision ( | ||||
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INH |
RSDb |
REc |
INH |
RSDb |
REc | ||
A | 3.0 | 2.96 | 1.24 | 0.89 | 3.07 | 2.34 | 2.12 |
6.0 | 6.11 | 1.84 | 1.03 | 6.12 | 2.00 | 2.53 | |
9.0 | 9.14 | 1.56 | 1.52 | 9.15 | 1.67 | 2.00 | |
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B | 1.0 | 1.01 | 1.02 | 1.92 | 1.03 | 3.00 | 1.85 |
1.5 | 1.47 | 2.01 | 1.15 | 1.54 | 2.67 | 1.27 | |
2.0 | 2.04 | 1.92 | 1.47 | 2.05 | 2.50 | 1.62 |
A selective study was performed to determine the effect of matrix on the absorbance by analyzing the placebo blank. In the analysis of placebo blank solution the absorbance in each case was equal to the absorbance of blank which revealed no interference. To assess the role of the inactive ingredients on the assay of INH, the general procedure was applied on the synthetic mixture extract by taking three different concentrations of INH within the range. The percentage recovery values were in the range 96.3–102.3% and 95.4–101.8% with RSD <4% indicating clearly the noninterference from the inactive ingredients in the assay of INH.
The robustness of the method was evaluated by making small incremental changes in the volume of reagent and reaction time, and the effects of the changes were studied by measuring the absorbance of the colored product. The changes had negligible influence on the results as revealed by small intermediate precision values expressed as %RSD (≤2.54%). Method ruggedness was demonstrated having the analysis done by three analysts and also by a single analyst performing analysis on three different instruments in the same laboratory. Intermediate precision values (%RSD) in both instances indicated acceptable ruggedness. These results are presented in Table
Method robustness and ruggedness expressed as intermediate precision.
Method | Nominal concentration | Robustness | Ruggedness | |||
---|---|---|---|---|---|---|
Reaction times* |
Volumes of FC reagent# | Volumes |
Interanalysts |
Interinstruments | ||
A | 3.0 | 1.45 | 1.69 | — | 2.20 | 2.32 |
6.0 | 1.72 | 1.35 | — | 1.74 | 1.92 | |
9.0 | 2.10 | 1.71 | — | 2.44 | 2.57 | |
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B | 1.0 | 1.72 | — | 1.59 | 1.78 | 2.12 |
1.5 | 1.84 | — | 1.61 | 2.11 | 1.62 | |
2.0 | 2.12 | — | 1.72 | 1.07 | 2.54 |
The proposed methods were applied to the quantification of INH in commercial tablets. The tablets were assayed by the official BP method [
Results of analysis of tablets by the proposed methods.
Tablets |
Label claim |
Found* (percent of label claim ± SD) | ||
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Official method | Proposed methods | |||
Method A | Method B | |||
Isokin-300 | 300 |
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Tabulated
Tabulated
To further assess the accuracy of the method, recovery experiment was performed by applying the standard-addition technique. The recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample. The test was done by spiking the preanalyzed tablet INH with pure INH at three different levels (50, 100, and 150%) of the content present in the preparation and the total was found by the proposed method. Each test was repeated three times. The recovery percentage values ranged between 99.28 and 101.7% with standard deviation in the range 0.99–1.32%. Closeness of the results to 100% showed the fairly good accuracy of the method. The results are shown in Table
Results of recovery study via standard addition method with tablet.
Method | Tablets studied | INH in |
Pure |
Total |
Pure INH recovered* |
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A | Isokin-300 | 2.96 | 1.5 | 4.41 |
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2.96 | 3.0 | 6.01 |
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2.96 | 4.5 | 7.56 |
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B | Isokin-300 | 1.01 | 0.5 | 1.51 |
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1.01 | 1.0 | 2.04 |
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1.01 | 1.5 | 2.55 |
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Two simple, rapid, selective, and sensitive methods have been proposed for the assay of INH in bulk drugs and in tablets. The methods are based on the well-characterized and established redox and complexation reactions and use very common and inexpensive chemicals and easily accessible instrument. The procedures described here are easily carried out and much simpler than the reported methods for INH (Table
Comparison of performance characteristics of the proposed methods with the existing methods.
Sl. No. | Reagent/s used | Methodology |
|
Linear range |
Remarks | References |
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1 | Epichlorohydrin | Measurement of purple colored coupled complex | 405 | 2–22 |
Longer reaction time and heating |
[ |
hydroxyphenacylchloride | 402 | 20–120 | ||||
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2 | Ethyl vanillin | Measurement of yellow colored hydrazone complex | 410 | 2–16 |
— | [ |
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3 | Vanillin | Measurement of yellow colored hydrazone complex | 405 | 1–12 | — | [ |
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4 | Isatin | Measurement of yellow colored hydrazone complex | 340 | 0–32 |
Requires close pH control and 40 min standing time, measurement at a shorter wavelength | [ |
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5 | Sodium 1,2-naphthoquinone-4-sulfonate and cetyltrimethyl ammonium bromide | Absorbance of condensation product measured | 500 | 2.0–5.6 | Employs a costly reagent | [ |
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6 | Chloranilic acid | Measurement of charge-transfer complex | 500 | 1.37–8.2 | Use of organic solvent, requires heating step |
[ |
Tetracyanoethylene | 480 | 6.85–34.27 | ||||
2,3-dichloro-5,6-dicyano-1,4-benzoquinone | 580 | 10.96–21.93 | ||||
Thymol blue | Measurement of ion-associate complex | 390 | 6.85–41.13 | Require close pH control, liquid-liquid extraction step | ||
Bromophenol blue | 410 | 1.37–6.85 | ||||
Bromocresol green | 320 | 1.37–8.22 | ||||
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7 | 2-Hydroxy-1,4-napthoquinone | Measurement of derivatized product | 365 | 5–25 | Requires close pH control, use of nonaqueous medium, measurement at a shorter wavelength | [ |
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8 | 1, 2-Naphthoquinone-4-sulfonate | Measurement of pink colored condensed product | 495 | 0.5–3.0 |
Requires close pH control | [ |
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9 | 6-Methyl-2-pyridinecarboxaldehyde | Measurement of hydrazone derivative | 328 | 2–16 | Measurement at lower analytical wavelength | [ |
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10 | 9-Chloroacridine | Measurement of absorbance of derivatized complex | 500 | — | Requires heating step, time consuming | [ |
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11 | 4,4′-Methylene-bis- |
Measurement of purple colored diazo-coupled complex | 495 | 0.1–15 |
Requires low temperature | [ |
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12 | 4,4′-Sulphonyldianiline | Measurement of purple colored diazo-coupled complex | 440 |
0.5–20 |
Requires low temperature | [ |
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13 | Tiron-NaIO4 | Measurement of red-colored oxidative-coupled product | 507 | 1.0–15 |
Use of multireaction system step | [ |
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14 | Tiron-KIO4 | Measurement of red-colored oxidative-coupled product | 505 | 1.5–18 |
Use of multistep reaction system | [ |
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15 | Uranyl acetate | Measurement of yellow-colored uranyl isonicotinoyl dithiocarbazate complex | 410 | — | Multistep reaction, time consuming | [ |
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16 | Neocuproine | Measurement of redox complex | 454 | 0.3–3.5 | Requires close pH control, a multistep reaction | [ |
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17 | Rose bengal | Measurement of acetone-chloroform extractable ion-pair complex | 555 | 2.8–5.6 | Requires time consuming and tedious extraction step, use of organic solvent | [ |
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18 | *NBS | Measurement of starch-iodine Complex | 572 | 0.1–3.4 | Multistep reaction, employs an unstable oxidant | [ |
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19 | *NBS | Absorbance of starch-iodine complex measured | 572 | 0.1–3.4 | Multistep reaction, employs an unstable oxidant | [ |
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20 | Potassium ferricyanide | Measurement of Prussian blue | 735 | 0.04–8 |
— | [ |
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21 | 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine | Measurement of redox complex | 558 | 0.62–6.15 | Multistep reaction, requires an expensive reagent | [ |
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The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors wish to thank the quality control manager, Cipla Ltd., Bangalore, India, for gifting pure isoniazid and the authorities of the University of Mysore, Mysore, for permission and facilities.