INTRODUCTION:

Isoniazid chemically isonicotinicacid hydrazide or pyridine-4-carboxylic acid hydrazide, show in figure 1, is pyridine carboxylic acid derivative.¹ It is first line drug which is commonly used in the treatment and prevention of tuberculosis. It is a prodrug and must be activated by a bacterial catalaseperoxidase enzyme in M. tuberculosis which is called KatG. KatG couples the isonicotinic acyl with NADH to form isonicotinic acyl-NADH complex. This complex binds tightly to the enoyl-acyl carrier protein reductase known as InhA, thereby blocking the natural enoyl- AcpM substrate and the action of fatty acid synthase. This process inhibits the synthesis of mycolic acid, required for the mycobacterial cell wall. A range of radicals are produced by KatG activation of isoniazid, including nitric oxide, -². It is rapidly absorbed and readily diffused into all fluids and tissues when it is given orally and it may be administered intramuscularly to critically ill patients.³ The possible adverse effects are rashes, hepatitis, abdominal liver function tests, sideroblastic anaemia, high anion gap acidosis, peripheral neuropathy, mild central nervous system effects.

The other reported severe side effects of fever and fatal liver damage. Hepatotoxicity can avoided the patient with close clinical monitoring, to be specific vomiting, nausea, loss of appetite and abdominal pain. The uses of isoniazid cause the peripheral neuropathy and CNS effects due to the pyridoxine depletion.Molecular formula of isoniazid is C₆H₇N₃O with a molecular mass 137.139g/mol.⁴ Freely soluble in water and sparingly soluble in alcohol.⁵ It can be used alone or in combination with acetyl Isoniazid, Rifampicin, Piperine, Indomethasin and pyrazinamide as an agent to treat tuberculosis. In the present review, we have complied the published analytical methods reported so far for the determination of isoniazid in pharmaceutical formulation and biological samples. Techniques like spectrophotometry, high performance liquid chromatography (HPLC), liquid chromatography – mass spectrophotometry, capillary gas chromatography have been used for analysis, from which HPLC methods are most extensively used. Overview of these methods for determination of isoniazid is shown in figure 2.

Fig. 1.Structure of isoniazid

Fig. 2. Over view of analytical methods for estimation of isoniazid in biological and pharmaceutical samples.

2. SOLUBILITY PREPARATION:

2.1 Solubility:

According to Biopharmaceutical Classification System (BCS) isoniazid is a class III drug (high solubility and low permeability). It was freely soluble in water and sparingly soluble in alcohol and slightly soluble in ether and in chloroform. The melting point of isoniazid is 171.7°C. ⁶

2.2 Sample preparation strategies:

Sample preparation is the integrated part of analytical methodology, and it was reported that approximately about 30% errors contributed from sample analysis was due to sample preparation.⁷ Various diluents used for the analysis of isoniazidinclude Methanol: Acetonitrile: buffer (2omM pH 2.5 heptanesulfonic acid sodium), water :methanol , 0.1 M phosphate buffer, (pH 5 Ortho phosphoric acid): methanol, 0.05 M ammonium acetate buffer (pH 6): acetonitrile and 10 mM Triethylamine (pH 10.5): acetonitrile. In major cases distilled water is used as solvent. The sample preparation technique for the extraction of isoniazid from the biological matrices like plasma, serum, urine, liver, kidney and brain was by deproteination with trichloroacetic acid, methanol and followed by solid phase extraction.

3. ANALYTICAL METHODS:

3.1 Spectrophotometry:

In the literature, 6 methods were reported for the estimation of isoniazid using spectrophotometry^8-14, of which 7 methods are for determining isoniazid alone, whereas the remaining are for quantifying isoniazid in combination with other drugs substance. Table 1 shows the summary of the reported spectroscopic methods indicating the basic principle, λ max, solvent and limit of detection (LOD).

3.2 Electrochemical methods:

Nada F Atta et al.¹⁵ describes the determination by electrochemical behaviour of isoniazid conventionally and economically using cyclic voltammetry, electrochemical impedance spectroscopy and linear response voltammetry. The authors used (PEDOT) Poly (3, 4-ethylenedioxy thiophene) electrode as sensor for the isoniazid in Briton –Robinson as buffer solution in absence and presence of CTAB and SDS and electro catalytic effects were found. Isoniazid give linear responses in the range of 0.1 to 8 μmol/ L and 10 to 100 μmol/L respectively, with correlation coefficients of 0.999 and 0.998 and detection limits were found to be 32nmol/L and 45nmol/L. The modified Electrode was utilized for determination of INH in human urine.

3.3 Chromatography;

3.3.1 HPLC:

3.3.1.1. Biological samples:

Various methods for the determination of isoniazid in biological samples like plasma, urine, biological fluids ^{16- 21}are listedin table 2.

3.3.1.2. Pharmaceutical samples:

Analytical methods for the determination of isoniazid in pharmaceutical dosage forms using HPLC ^22-26are shown in table 3.

Table 1. Representative Spectrophotometric methods for analysis of isoniazid

Compounds	Method	λ max	Solvent	LOD	Ref.
Isoniazid	Spectrophotometric method	405	3%Vanillin, 0.5 Methanolic Hydrochloric acid	-	8
Isoniazid, Rifampicin, Piperine	Simultaneous absorption correction method	263, 338, 477	Methanol, distilled water	0.318	9
Isoniazid, Pyridoxine	Simultaneous equation method	263, 292	Distilled water	0.481	10
Isoniazid, Rifampicin	Simultaneous equation method	263, 337	Ethanol	0.585	11
Isoniazid, Ethambutol HCl	Spectrophotometric method	572, 310, 218	Iodine – starch solution, Hydroquinone solution	-	12
Isoniazid, Rifampicin	Spectrophotometric method	264,474	Distilled water	-	13
Isoniazid	Spectrophotometric method	364	Double distilled water	0.1	14

Matrix	IS	Sample preparation	Mobile phase	Column	Detection	λ max (nm)	Flow rate (ml/min)	LOD/ LLOQ (μg/ml)	Ref
Heparinised plasma	-	Deproteinated with 40μL 15% (w/v) trichloroacetic acid	1-hexanesulfonic acid sodium salt solution (20mM pH 3, phosphoric acid):Acetonitrile (90:10 v/v)	Octade-cylsilane-bonded silica 150 mm × 3 mm, 3.5-μm	UV	290	0.4	0.5	16
Human plasma	Terramycin	-	Methanol: Acetonitrile: buffer (2omM pH 2.5 heptanesulfonic acid sodium) (10:8:82 v/v/v).	Synergi Max-RP C12 (250x 4.6 mm, 4 μm )	UV	264	2	0.023	17
Human urine	-	-	water :methanol (85:15v/v)	Merck C8, (250 x 4.6mm, 5 μm)	UV	274	1.2	-	18
Rat plasma, brain, liver, kidney	-	Deprotinizing agent using methanol	0.1 M phosphate buffer,( pH 5 Ortho phosphoric acid) : methanol (50:50 v/v)	Waters, Symmetry shield RP- 18, 150 x 4.6mm cm, 5 μm)	UV	254	0.9	0.150	19
Human plasma	Nicotinamide	Deproteinated by trichloroacetic acid.	0.05 M ammonium acetate buffer (pH 6): acetonitrile (99:1, v/v).	Pinnacle II C18 (150 x 4.6 mm, 5μm)	UV	275	1.2	-	20
Human serum	2-pyridylamine	Solid phase extraction	10 mM Triethylamine (pH 10.5): acetonitrile (67:33, v/v)	Octasilil C8 (Purospher RP8) of the 250 x 4.6 mm, 5 μm)	UV	260	1	5.16	21

Table 3 Reported analytical methods for determination of isoniazid with other drugs like Isoniazid, Rifampicin, Piperine, Pyrazinamide, Ethambutol Hydrochloride, pyridoxine hydrochloride and Indomethasin

Study aim	Mobile phase	Column	Detection	λ max (nm)	Flow rate (ml/min)	LOD (μg/ ml)	Ref
Simultaneous determination of Isoniazid, Rifampicin, Piperine	Sol. A Water+0.1% acetic acid buffer, 2.5mMammonium acetate (10%) Sol. B Acetonitrile +0.1% acetic acid buffer (90%)	E-Merck RP-18(250 x 4.0mm, 5 μm)	UV	263	0.4	0.167	22
Simultaneous determination of Isoniazid, Pyrazinamide, Indomethasin	Water: Methanol: Tetra hydro furan ( 59:39:2 v/v)	YMC-ODS (150 x 4.6mm)	UV	328	2	-	23
simultaneous determination of Ethambutol Hydrochloride, Isoniazid	Methanol: ammonium acetate buffer (pH-7.03) (50:50).	C18 Thermo Hypersil ODS, (250 x 5.4 mm, 4.5μm)	UV	276	1.3	0.569	24
simultaneous determination of Isoniazid, Ethambutol Hydrochloride,	potassium dihydrogen orthophosphate buffer pH 6.9	Inertsil ODS 3 V (250 x 4.6 mm, 5 μm)	UV	247	1.5	0.083	25
simultaneous determination of pyridoxine hydrochloride, isoniazid, pyrazinamide, Rifampicin	Acetonitrile (A) 15mmol L.1 potassium dihydrogen phosphate buffer pH 4.0, O-phosphoric acid (B). A:B (50:50v/v)	250 × 4.6 mm I.D. C18 column packed with 5 mm	UV	235	1	0.063	26

3.4 GC:

Mohammad Yar Khuhawar et.al.²⁷developed a GC method for determination of isoniazid and hydrazine in pharmaceutical formulation. After free column derivatization with Ethyl chloroformate. Phenyl hydrazine (PHZ) was used as an internal standard. They carried out separation on HP-5 column (30 m x 0.32 mm I.D). Flame ionization detector is used for the detection of samples. With initial column temperature of 150˚C for 1 min, to 250˚C increased the temperature at a heating rate of 10˚C/min. Nitrogen gas supplied with a flow rate of 4ml/min. The linearity of the calibration curve was found to be between 3.5 – 37.5 mg/ml. The validation studies were performed and the limit of detection was found to be 0.18ng/mL. Capillary gas chromatography method was developed by M.Y. Khuwar et. al.²⁸for determination of isoniazid and hydrazine in pharmaceutical formulation and blood in the presence of Phenyl hydrazine (IS) after derivitazing the pre column with trifluoroacetone. The samples were completely separated on a HP-5(30*0.32mm I.D) column and detect samples with flame ionization dectector. Initially the column temperature was maintained at 100°C, with a heating rate of 30°C/min increased up to 280°C. The run time was 7min. Nitrogen flow rate was 1mL/min. The linearty range of the proposed method was 2.5-25μg/mL. Isoniazid LOD was found to be62.5pg.

3.5 LC-MS:

LC-MS/MS method was developed by P Pavan Kumar et. al.²⁹ for simultaneous determination of isoniazid and ethambutol in dried blood spots in which Nicorandil was used as internal standard. Punched 3mm spotted and dried blood Samples were extracted with methanol. The samples were separated on a kromasil C18 column by using a 35:65 v/v mixture of 0.1% formic acid in water and methanol as a mobile phase at a flow rate 0.8ml/ min. The linearity range of the proposed method was 100-500ng/mL for isoniazid and was successfully applied to human pharmacokinetic study.

3.6 HPTLC:

Shajahan Puthusseri et.al.³⁰ developed an HPTLC method for isoniazid for simultaneous estimation of isoniazid, pyridoxine hydrochloride and Rifampicin in combined pharmaceutical dosage form. They carried out separation on Aluminium plates precoated with silica gel 60 G F254 using the mixture of Ethyl acetate: Methanol: Acetone: Acetic acid (5.5: 2.0: 2.0: 0.5, v/v) as the mobile phase. The detection of spot was carried out by using UV detector at 254nm, the evaluation of separated zones were performed. The linearity of the calibration curve was found to be between 200-1000ng/spot.

4. CHALLENGES:

Isoniazid as classified under the Biopharmaceutical Classification System class III; the drug has high solubility and low permeability. Owing to its lower solubility in aqueous solution majority of methods developed for isoniazid using chromatographic techniques included organic phase for the preparation of the stock solution and further dilutions were made in mixture of solvents. The solution stability of drug in hydro organic solvents for isoniazid was less than 24hours which indicate the degradative property of drug in solution form and calls a need for a rapid analytical technique for the estimation of isoniazid particularly in electrophoretic technique which requires longer run times and repeated analysis for obtaining precise results.

5. CONCLUSION:

A large number of techniques are available for the estimation of isoniazid in pharmaceutical formulations and biological samples. The survey of analytical data revealed that HPLC methods are predominant for the estimation of drug alone or in combination with other drugs in various formulation types. So for the precise and accurate separation of isoniazid in various formulations recommended method of analysis includes HPLC with UV detector as it provides faster analysis time and has more separation selectivity than most other available techniques. This review carried out an overview of the state-of-art analytical methods for the determination of isoniazid in different formulations.

6. ACKNOWLEDGMENT:

The authors wish to thanks Mr. A. Mohana Krishna, Assistant Professor, Sree Vidyanikethan College of Pharmacy, Tirupati, Andhra Pradesh, India for his constant encouragement and is thankful to the Department of Pharmaceutical Analysis, Sree Vidyanikethan College of Pharmacy for providing facility for the work.

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Received on 10.02.2015 Accepted on 25.03.2015

Asian J. Pharm. Ana. 5(1): Jan.- March 2014; Page 41-45

DOI: 10.5958/2231-5675.2015.00008.3