REVIEW: AVAILABLE ANALYTICAL METHODS FOR THE ESTIMATION OF FIRST LINE, SECOND LINE ORAL AND NEWER ANTI-TB DRUGS

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REVIEW: AVAILABLE ANALYTICAL METHODS FOR THE ESTIMATION OF FIRST LINE, SECOND LINE ORAL AND NEWER ANTI-TB DRUGS

Riddhi J. Jani ^* and Paresh U. Patel

Department of Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Mehsana-Gozaria Highway, Ganpat Vidyanagar, Mehsana - 384012, Gujarat, India.

ABSTRACT: Tuberculosis (TB) is one of the top ten causes of death worldwide. Presently, one-quarter of the world's population is thought to be infected with TB. New infections occur in about 1% of the population each year. Tuberculosis is a major global health threat. There is a progressive increase in multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR). Multi drug resistance (MDR)-TB and Extensively drug resistance (XDR)-TB poses a vital challenge to the control of tuberculosis. Numbers of drugs are available in the market for the treatments of tuberculosis as well as many new drugs are also available for the treatment of MDR-TB and XDR-TB. This review article covers most of the different official and reported analytical methods for the estimation of the first line, second line oral, and newer anti-TB drugs. The main objective of this review is to classify, summarize, and discusses the different proposed analytical methods for the estimation of above mentioned anti-TB drugs alone and in combination with other drugs in bulk, pharmaceutical formulation and biological matrices.

Keywords:

Bedaquiline, Delamanid, Pretomanid, RP-HPLC, HPTLC, LC-MS

INTRODUCTION: Tuberculosis (TB) is the most important airborne infectious disease caused by a bacterium called Mycobacterium tuberculosis (MTB). The one third of the world population is the infected by mycobacterium tuberculosis according to the World Health Organisation (WHO) estimation. HIV infected persons, immigrants from countries with high rates of tuberculosis, the homeless, healthcare professionals, intravenous drug users, a person taking immunosuppressive agents and those in an institutional setting such as nursing homes and correctional facilities group at high risk for tuberculosis infection there is a progressive increase in multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR).

Anti-Tb drugs are classified as:

(i) Oral first-line drugs and extended first-line drugs (Isoniazid, Rifampicin, Ethambutol, Pyrazin-amide, Rifabutin, Rifapentine), (ii) Injectable anti-TB drugs (Streptomycin, Kanamycin, Amikacin, Capreomycin, Viomycin) (iii) Fluoroquinolones (Ciprofloxacin, Ofloxacin, Levofloxacin, Moxi-floxacin, Gatifloxacin) (iv) Oral second-line anti-TB drugs (Ethionamide/Prothionamide, Cycloserine, Terizi-done, Para-aminosalicylic acid) (v) Anti-TB drugs with limited data on efficacy and long-term safety in the treatment of drug-resistant TB (This group includes new anti-TB agents) (Bedaquiline, Delamanid, Pretomanid, Linezolid, Clofazimine, Amoxicillin / clavulanate, Imipenem / cilastatin, Meropenem, High-dose Isoniazid, Thioacetazone, Clarithromycin).

The increasing interest in the oral first line and the oral second line as well as new anti-TB drugs like bedaquiline, delamanid, and pretomanid led us to review the official and reported analytical methods for the estimation of these anti-TB drugs alone and in combination with other drugs in bulk, pharmaceutical formulation and biological matrices.

TABLE 1: DRUG PROFILE OF ANTI-TB DRUGS (ORAL FIRST LINE, ORAL SECOND LINE AND NEWER ANTI-TB DRUGS)

Drug	Chemical Structure	Chemical Name	Chemical Formula	pKa	Log P
Isoniazid		4-Pyridine-carboxylic acid hydrazide	C₆H₇N₃O	1.82	-0.8
Rifampicin		(2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-1,2-dihydro-5,6,9,17,19-pentahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-(4-methylpiperazin-1-yliminomethyl)-1,11-dioxo-2,7-(epoxypentadeca-1,11,13-trienoimino)naphtho[2,1-b]furan-21-yl acetate	C₄₃H₅₈N₄O₁₂	1.7	2.7
Ethambutol		2,2'-(1,2-Ethanediyldiimino)-bis-1-butanol	C₁₀H₂₄N₂O₂	9.49	-0.3
Pyrazinamide		Pyrazinecarboxamide or Pyrazine-2-carboxamide	C₅H₅N₃O	0.5	-0.6
Rifabutin		9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,22E,24Z)-6,16,18,20-Tetrahydroxy-1'-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethylspiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-furo[2',3':7,8]naphth[1,2-d]imidazole-2,4'-piperidine]-5,10,26-(3H,9H)-trione-16-acetate	C₄₆H₆₂N₄O₁₁	6.9	4.5
Rifapentine		(2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-8-{(E)-[(4-cyclopentylpiperazin-1-yl)imino]methyl}-5,6,9,17,19-pentahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-1,11-dioxo-1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienoimino)naphtho[2,1-b]furan-21-yl acetate	C₄₇H₆₄N₄O₁₂	-1.6	4
Ethionamide		2-Ethyl-4-pyridinecarbothioamide	C₈H₁₀N₂S	4.49	0.5
Cycloserine		D-4-Amino-3-isoxazolidinone	C₃H₆N₂O₂	4.4, 7.4	-0.9
Terizidone		4-[({4-[N-(3-oxo-1,2-oxazolidin-4-yl)carboximidoyl]phenyl}methylidene)amino]-1,2oxazolidin-3-one	C₁₄H₁₄N₄O₄	3.54	0.17
p-aminosalicylic acid		4-Aminosalicylic acid or 4-Amino-2-hydroxybenzoic acid	C₇H₇NO₃	3.25	1.6
Bedaquiline		(1R,2S)-1-(6-bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-2-naphthalen-1-yl-1-phenylbutan-2-ol	C₃₂H₃₁BrN₂O₂	1.57	7.25
Delamanid		(2R)-2-methyl-6-nitro-2-[(4-{4-[4-(trifluoromethoxy)phenoxy]-1- piperidinyl}phenoxy)methyl]-2,3-dihydroimidazo[2,1-b][1,3]oxazole	C₂₅H₂₅F₃N₄O₆	5.51	6.14
Pretomanid		6S)-2-nitro-6-{[4-(trifluoromethoxy)phenyl]methoxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine	C₁₄H₁₂F₃N₃O₅	-3	4.14

Analytical Methods for Anti-TB Drugs: Different official and reported analytical methods such as UV-visible spectrophotometric, Spectro-fluorometric, High-performance liquid chromato-graphy (HPLC), High-performance thin-layer chromatography (HPTLC), Gas chromatography (GC), Micellar electro-kinetic capillary chromato-graphy, Electrochemical, Titrimetric, Liquid chromatography / Mass spectrometry (LC/MS), Capillary-electrophoresis, Flow injection analysis, Chemi-luminescence, etc. are available for estimation of the first line, oral second line and newer anti-TB drugs in bulk, pharmaceutical formulation and biological matrices.

A review of different official and reported analytical methods is listed as follow:

Official Analytical Methods:

TABLE 2: UV-VISIBLE SPECTROPHOTOMETRIC METHODS

S. no.	Drug / Sample	Pharmacopoeia	Solvent	Wavelength of Detection	Ref. no.
1	Cycloserine Tablet	IP 2018	Water, 0.2 M NaOH, 1 M acetic acid, sodium nitroprusside solution	625 nm	29
2	Ethionamide & Ethionamide Tablets	USP 2013	Methanol	290 nm	30
3	Rifampicin, Rifampicin Capsule & Rifampicin Oral Suspension	BP 2016	Methanol, phosphates buffer pH 7.4	475 nm	31
4	Pyrazinamide Tablets	IP 2018 BP 2016	Water	268 nm	49 32
5	Rifampicin	EP 2008	Methanol, phosphates buffer pH 7.4	475 nm	33

TABLE 3: LIQUID CHROMATOGRAPHIC METHODS

S. no.	Drug / Sample	Pharmacopoeia	Column	Mobile phase	Flow Rate (ml/ min)	Wavelength of Detection	Ref. no.
1	Isoniazid & Isoniazid Tablets	IP 2018	ODS (15 cm × 4.6 mm, 5 µm)	A mixture of a solution prepared by dissolving 1.4 g disodium hydrogen phosphate and 1 ml of triethylamine to 1000 ml with water (pH 6.0 adjusted by OPA) & Acetonitrile (96:4, v/v)	1	265 nm	34
2	Rifampicin, Rifampicin Capsules, Rifampicin Oral Suspension &Rifampicin Tablets	IP 2018	Octylsilane (10 cm × 4.6 mm, 5 µm)	A mixture of a solution containing 0.1% v/v of OPA, 0.19% w/v of sodium perchlorate, 0.59% w/v of citric acid &2.09% w/v of potassium dihydrogen phosphate & Acetonitrile (65:35, v/v)	1.5	254 nm	35
3	Rifampicin and Isoniazid Tablets	IP 2018	ODS (25 cm × 4.6 mm, 5 µm)	A. A mixture of a buffer solution pH 6.8 prepared by dissolving 1.4 g disodium hydrogen orthophosphate anhydrous in 1000 ml of water (pH 6.8 ± 0.05 adjusted by dil. phosphoric acid) & Acetonitrile (96:4, v/v) B. A mixture of the buffer solution and Acetonitrile (45:55, v/v) Using mixture, A & B ingradient programme	1.5	238 nm	35
4	Rifampicin, Isoniazid and Ethambutol Tablets 1) For Rifampicin and Isoniazid Tablets 2) For Ethambutol Hydrochloride	IP 2018	1. ODS (25 cm × 4.6 mm, 5 µm) 2. Zorbax SB CN (15 cm × 4.6 mm, 5 µm)	1) A. A mixture of a buffer solution pH 6.8 prepared by dissolving 1.4 g disodium hydrogen orthophosphate anhydrous in 1000 ml of water (pH 6.8 ± 0.05 adjusted by dil. phosphoric acid) & Acetonitrile (96: 4, v/v) B. A mixture of the buffer solution and Acetonitrile (45:55, v/v) Using mixture, A & B in gradient elution programme 2) A mixture of Acetonitrile & buffer solution pH 7.0 prepared by dissolving 1 ml of triethylamine in 1000 ml of water (pH 7.0 adjusted by dil. phosphoric acid (50:50, v/v)	1) 1.5 2) 1	1) 238 nm 2) 200 nm	35
5	Rifampicin, Isoniazid and Pyrazinamide Tablets	IP 2018	ODS (25 cm × 4.6 mm, 5 µm)	A. A mixture of a buffer solution pH 6.8 prepared by dissolving 1.4 g disodium hydrogen orthophosphate anhydrous in 1000 ml of water (pH 6.8 ± 0.05 adjusted by dil. phosphoric acid) & Acetonitrile (96: 4, v/v) B. A mixture of the buffer solution and Acetonitrile (45:55, v/v) Using mixture, A & B in gradient programme	1.5	238 nm	35
6	Rifampicin, Isoniazid, Pyrazinamide and Ethambutol Tablets 1) For Rifampicin Isoniazid and pyrazinamide Tablets 2) For Ethambutol Hydrochloride	IP 2018	1) ODS (25 cm × 4.6 mm, 5 µm) 2) Zorbax SB CN (15 cm × 4.6 mm, 5 µm)	1) A. A mixture of a buffer solution pH 6.8 prepared by dissolving 1.4 g disodium hydrogen orthophosphate anhydrous in 1000 ml of water (pH 6.8 ± 0.05 adjusted by dil. phosphoric acid) & Acetonitrile (96: 4, v/v) B. A mixture of the buffer solution and Acetonitrile (45:55, v/v) Using mixture, A & B in gradient programme 2) A mixture of Acetonitrile & buffer solution pH 7.0 prepared by dissolving 1 ml of triethylamine in 1000 ml of water (pH 7.0 adjusted by dil. Phosphoric acid) (50:50, v/v)	1) 1.5 2) 1	1) 238 nm 2) 200 nm	35
7	Ethambutol hydrochloride, Ethambutol Injection & Ethambutol Tablets	IP 2018	Zorbax SB CN (15 cm × 4.6 mm, 5 µm)	A mixture of buffer solution prepared by dissolving 1 ml of triethylamine in 1000 ml of water (pH 7.0 adjusted by orthophosphoric acid) & Acetonitrile (50:50, v/v)	1	200 nm	36
8	Ethambutol & Isoniazid Tablets 1) For Isoniazid 2) For Ethambutol	IP 2018	1) ODS (15 cm × 4.6 mm, 5 µm) 2) Zorbax SB CN (15 cm × 4.6 mm, 5 µm)	1) A mixture of buffer solution pH 6.8 prepared by dissolving 1.4 g disodium hydrogen phosphate in 1000 ml of water (pH 6.8 ± 0.05 adjusted by dil. phosphoric acid) & Acetonitrile (96:4, v/v) 2) A mixture of buffer solution prepared by dissolving 1 ml of triethylamine in 1000 ml of water (pH 7.0 ± 0.05 adjusted by phosphoric acid) & Acetonitrile (50:50, v/v)	1	1) 254 nm 2) 200 nm	36
9	Ethionamide & Ethionamide Tablets	IP 2018	ODS (25 cm × 4.6 mm, 5 µm)	A mixture of buffer solution prepared by dissolving 2 ml of triethylamine in 1000 ml of water (pH 6.0 adjusted by orthophosphoric acid) & Acetonitrile (60:40, v/v)	1	290 nm	37
10	Cycloserine & Cycloserine Capsules	IP 2018	Octylsilane (25 cm × 4.6 mm, 5 µm)	0.1% w/v of methane sulphonic acid & 0.78% w/v potassium dihydrogen orthophosphate in water (pH 6.0 adjusted by dil. NaOH)	1	227 nm	29
11	Isoniazid Injection & Isoniazid Tablets	JP 2006	ODS (15 cm × 4.6 mm, 5 µm)	Dissolve 6.80 g of potassium dihydrogen phosphate in water to make 1000 ml. Separately, to 5.76 g of phosphoric acid add water to make 1000 ml. Mix these solutions to make a solution having pH 2.5. To 500/400 ml of this solution, add 500/600 ml methanol & add 2.86g of sodium tridecanesulfonate to dissolve.	-	265 nm	38
12	Rifampicin & Rifampicin Capsules	JP 2006	ODS (10 cm × 4.6 mm, 5 µm)	Dissolve 4.2 g of citric acid monohydrate and 1.4 g of sodium perchlorate in 1000 ml of a mixture of water, acetonitrile and phosphate buffer solution, pH 3.1 (11:7:2, v/v/v)	-	254 nm	39
13	Isoniazid & Isoniazid Injection	USP 2013	ODS (25 cm × 4.6 mm, 1.5 to 10 μm)	Dissolve 4.4. g of docusate sodium in 600 ml of methanol, add 400 ml of water, (pH 2.5 adjusted by 2N sulfuric acid)	1.5	254 nm	40
14	Isoniazid Tablets	USP 2013	ODS (30 cm × 3.9 mm, 1.5 to 10 μm)	Buffer: Methanol (95:5, v/v) Buffer solution: Prepare a 0.1 M monobasic potassium phosphate solution, adjust with 10 N NaOH to pH of 6.9, add sufficient triethanolamine to obtain a solution having a known concentration of 0.2 mM of triethanolamine & mix	1.5	254 nm	40
15	Rifabutin, Rifabutin Capsules & Rifabutin Oral Suspension	USP 2013	Octylsilane (12.5 cm × 4.6 mm, 5 μm) -For Rifabutin oral suspension Octylsilane (15 cm × 4.6 mm, 5 μm)	A mixture of acetonitrile & 0.1 M monobasic potassium phosphate (pH 6.5 ± 0.1 adjusted by 2N NaOH) (50:50, v/v)	1	254 nm	41
16	Rifampicin, Rifampicin Capsules, Rifampicin for Injection & Rifampicin Oral Suspension	USP 2013	Octylsilane (10 cm × 4.6 mm, 5 μm)	A mixture of water, acetonitrile, phosphate buffer, 0.1 M citric acid & 0.5 M sodium perchlorate (510:350:100:20:20, v/v/v/v/v) For Rifampicin Oral Suspension: (500:360:100:20:20, v/v/v/v/v)	1.5	254 nm	42
17	Rifampicin and Isoniazid Capsules, Rifampicin Isoniazid and Pyrazinamide Tablets, Rifampicin, Isoniazid, Pyrazinamide & Ethambutol Hydrochloride Tablets	USP 2013	ODS (25 cm × 4.6 mm, 5 µm) -For Ethambutol HCl: CN (15 cm × 4.6 mm, 5 µm)	Buffer solution- Dissolve 1.4 g of dibasic sodium phosphate in 1L of water (pH 6.8 adjusted by phosphoric acid) Solution A: Buffer solution & Acetonitrile (96:4, v/v) Solution B: Buffer solution & Acetonitrile (45:55, v/v) Use variable mixtures of solution A & B in gradient elution programme - For Ethambutol Hydrochloride: Buffer solution: Mix 1.0 ml of triethylamine and 1 L of water (pH 7 adjusted by phosphoric acid) Use mixture of Acetonitrile & Buffer solution (50:50, v/v)	1.5 1	238 nm 200 nm	42
18	Pyrazinamide Oral suspension	USP 2013	Octylsilane (25 cm × 4.6 mm, 5 μm)	Acetonitrile and solution of 10 mM monobasic sodium phosphate (pH 3.5 adjusted by phosphoric acid) (10:90, v/v)	0.8	215 nm	43
19	Pyrazinamide Tablets	USP 2013	ODS (15 cm × 3.9 mm, 1.5 to 10 μm)	Prepare pH 8.0 phosphate buffer (pH 3.0 adjusted by phosphoric acid). Mix 10 ml of acetonitrile with 1 L of this solution.	1	270 nm	43
20	Ethambutol Hydrochloride Tablets	USP 2013	CN (15 cm × 4.6 mm, 5 µm)	Acetonitrile & Buffer solution prepared by mixing 1.0 ml of triethylamine and 1 L of water (pH 7 adjusted by phosphoric acid) (1:1, v/v)	1	200 nm	44
21	Amino Salicylic Acid & Amino Salicylic Acid Tablets	USP 2013	ODS (25 cm × 4.6 mm, 1.5 to 10 μm)	Mixture of 0.05 M dibasic sodium phosphate, 0.05 m monobasic sodium phosphate & methanol containing 1.9 g of tetrabutyl-ammonium hydroxide (425:425:150, v/v/v)	1.5	254 nm	45
22	Cycloserine & Cycloserine Capsules	USP 2013	ODS (25 cm × 4.6 mm, 5 µm)	Dissolve 0.5 g of sodium 1-decanesulfonate in 800 ml water, add 50 ml of acetonitrile & 5 ml of glacial acetic acid (pH 4.4 adjusted by 1 N NaOH)	1	219 nm	46
23	Rifabutin	BP 2016	Octylsilyl (0.110 m × 4.6 mm, 5 µm)	Acetonitrile & a 13.6 g/ml solution of potassium dihydrogen phosphate (pH 6.5 adjusted by dil. NaOH)	1	254 nm	47
24	Rifabutin	EP 2008	Octylsilyl (0.110 m × 4.6 mm, 5 µm)	Acetonitrile & a 13.6 g/ml solution of potassium dihydrogen phosphate (pH 6.5 adjusted by dil. NaOH)	1	254 nm	48

TABLE 4: TITERIMETRIC METHOD

S. no.	Drug / Sample	Pharmacopoeia	Description	Ref. no.
1	Pyrazinamide	IP 2018	Weigh 0.3 g Pyrazinamide & transfer to the flask of ammonium distillation apparatus. Add 200 ml of water & 75 ml NaOH solution. Boil & collecting the distillate in 50 ml of 0.05 M sulphuric acid. Boil to the complete distillation of the ammonia and titrate the excess of acid with 0.1 M NaOH, using methyl red as an indicator.	49
2	Isoniazid Oral Solution	USP 2013	Isoniazid Oral Solution in 50 ml of a mixture of 1 part of KBr in 10 parts of dil. HCl. Proceed as per Nitrite Titration.	40
3	Pyrazinamide	USP 2013	Place about 300 mg of Pyrazinamide in 500 ml Kjeldahl flask, dissolve in 100 ml of water & add 75 ml of 5 N NaOH. Connect the flask to the condenser, the delivery tube of which dips into 20 ml of boric acid solution. Boil vigorously to complete the distillation of the ammonia. After cooling, add methyl purple & titrate with 0.1 N HCl.	43
4	Ethambutol Hydrochloride	USP 2013	Add 200 mg of Ethambutol Hydrochloride in a mixture of 100 ml of glacial acetic acid & 5 ml of mercuric acetate. Add crystal violet. Titrate with 0.1N perchloric acid. The color change at the endpoint is from blue to blue-green.	44
5	Isoniazid	BP 2016 EP 2008	Dissolve 0.250 g Isoniazid in water and dilute 100 ml with water. To 20 ml of the solution, add 100 ml of water, 20 ml HCl, 0.2 g of KBr, and 0.05 ml methyl red solution. Titrate with 0.0167 M potassium bromate until the red color disappears.	50 51
6	Isoniazid Injection	BP 2016	Dilute 0.4 g of Isoniazid to 250 ml with water. To 25 ml of the solution, add 25 ml of 0.05 M Br & 5 ml HCl. Allow standing for 15 min. Add 1 g of KI & titrate with 0.1 M sodium thiosulphate using starch mucilage as an indicator.	50
7	Isoniazid Tablets	BP 2016	Dissolve a quantity equivalent to 0.4 g of Isoniazid and dilute 100 ml with water. To 50 ml of the solution, add 50 ml of water, 20 ml HCl, 0.2 g of KBr, and titrate with 0.0167 M potassium bromate. Determining the end-point electrometrically.	50
8	Pyrazinamide	BP 2016 EP 2008	Dissolve 0.100 g Pyrazinamide in 50 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end point potentiometrically.	32 52
9	Ethambutol Hydrochloride	BP 2016 EP 2008	Dissolve 0.200 g of Ethambutol Hydrochloride in 50 ml of water & add 1 ml of 0.1 M HCl. Carry out a potentiometric titration, using 0.1 M NaOH.	53 54
10	Ethambutol Tablets	BP 2016	Add 20 ml of 2 M NaOH to a quantity of tablet powder equivalent to 0.2 g of Ethambutol Hydrochloride. Extract with three successive 25 ml quantities of a mixture of 3 volumes of chloroform and 1 volume of propan-2-ol. Filter each extract. Add 100 ml anhydrous acetic acid to the combined extracts & carry out non-aqueous titration, using 1-naphtholbenzein solution as an indicator.	53
11	Ethionamide	BP 2016 EP 2008	Dissolve 0.150 g of Ethionamide in 50 ml anhydrous acetic acid. Titrate with 0.1 M perchloric acid, determining the end point potentiometrically.	55 56
12	Isoniazid	JP 2006	Dissolve 0.3 g Isoniazid in 50 ml acetic acid & 10 ml of acetic anhydride. Titrate with 0.1 mol/L perchloric acids, until the color of the solution changes from yellow to green.	38
13	Pyrazinamide	JP 2006	Dissolve 0.1 g Pyrazinamide in 50 ml of acetic anhydride. Titrate with 0.1 mol/L perchloric acid, determining the end point potentiometrically.	57
14	Ethambutol Hydrochloride	JP 2006	Dissolve 0.2 g Ethambutol Hydrochloride in 20 ml of water & add 1.8 ml copper (II) sulfate. Add 7 ml of NaOH with shaking, add water to make 50 ml. To 10 ml of this solution, add 10 ml of ammonia-ammonium chloride buffer of pH 10.0 and 100 ml water. Titrate with 0.1 mol/L disodium dihydrogen ethylenediamine tetraacetate until the color of the solution changes from blue-purple to light yellow.	58
15	Ethionamide	JP 2006	Dissolve 0.3 g Ethionamide in 50 ml of acetic acid. Titrate with 0.1 mol/L perchloric acids, until the color of the solution changes from orange-red to dark orange-brown, using 2 ml p-naphtholbenzein solution as an indicator.	59

Reported Analytical Methods:

UV-Visible Spectrophotometric Methods:

TABLE 5: UV-VISIBLE SPECTROPHOTOMETRIC METHODS

S. no.	Drug / Sample	Method	Solvent	Linearity Range μg/ml	Wavelength of Detection	Ref. no.
Isoniazid
1	Isoniazid in urine	Spectrophotometric (Based on the formation of an orange, yellow colour complex between isoniazid and ammonium metavanadate in an acid medium)	Distilled water	1.37-13.70	420 nm	60
2	Isoniazid in pharmaceuticals	Spectrophotometric (Using Cerium (IV) and Two Acid Dyes) Method A (using methyl orange) Method B (using indigo carmine)	Distilled water	For Method A: 0.3–3.0 For Method B: 0.5–7.0	For Method A: 520 nm For Method B: 610 nm	61
3	Isoniazid in presence of Rifampicin in pharmaceuticals & urine	Spectrophotometric (using isatin as a reagent)	Distilled water	0-32	340 nm	62
4	Isoniazid in pure & pharmaceutical formulation	Spectrophotometric (Based on the oxidation of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) by sodium metaperiodate (SPI) followed by oxidative coupling with INH in an alkaline medium.	Distilled water	1-15	507 nm	63
5	Isoniazid in pharmaceuticals	Spectrophotometric (Redox-Reaction Based) Method A (using Folin-Ciocalteu reagent) Method B (using iron (III) and ferricyanide)	Water	For Method A: 0.5–10.0 For Method B: 0.2–3.0	760 nm	64
6	Isoniazid in pharmaceuticals	Spectrophotometric (Using 6,7- dichloroquinoline-5,8-dione)	Water & Ethanol	2–25	645 nm	65
7	Isoniazid in pharmaceuticals	Spectrophotometric (Using Natural Aldehyde like cis-cinnamaldehyde)	Distilled Water	0.5-2.5	364 nm	66
8	Isoniazid in pharmaceuticals	Spectrophotometric (Using its Schiff’s base derivatives)	Methanol	0.25-5	421 nm	67
9	Isoniazid in bulk & pharmaceuticals	Visible Spectrophotometric (based on the formation of yellow colored chromogen with ethanolic p-dimethylamino benzaldehyde solution in the presence of conc. HCl)	Ethanol	100-600	395 nm	68
10	Isoniazid in pure & pharmaceutical formulation	Spectrophotometric (Using vanillin)	0.5M ethanolic HCl acid	1‐12	405 nm	69
11	Isoniazid in Tablets	Colorimetric	Distilled water	3-18	530 nm	70
12	Isoniazid in bulk & pharmaceutical dosage forms.	Colorimetric (using ethyl vanillin in presence of 0.5M NaOH)	Distilled Water	2-16	410 nm	71
Isoniazid in combination with other drugs
13	Isoniazid and Pyridoxine in tablet dosage form	Spectrophotometric (Simultaneous Equation Method)	Distilled Water	5-25	Isoniazid: 263 nm Pyridoxine: 290 nm	72
14	Isoniazid (INH) and Ritodrine Hydrochloride (RTH) in pure dosage forms	Spectrophotometric (Based on the diazotisation of 4,4’-sulphonyldianiline (dapsone, DAP) followed by a coupling reaction with either INH or RTH in sodium hydroxide medium)	Deionised water	Isoniazid: 0.5-20 Ritodrine HCl: 0.5-18	Isoniazid: 440 nm Ritodrine HCl: 460 nm	73
15	Isoniazid and Lamivudine in marketed formulations	Spectrophotometric (Q-absorption ratio)	Phosphate buffer (pH7.4)	5-30	Iso-absorptive point: 246 nm Second wavelength: 272 nm	74
16	Isoniazid and Pyridoxine HCl in commercial tablets	Spectrophotometric (Area under curve)	Methanol	Isoniazid: 5-15 Pyridoxine HCl: 6–18	Isoniazid: 262.2-272.2 nm Pyridoxine HCl: 289.8-299.8 nm	75
17	Isoniazid and Rifampicin from pharmaceutical preparations and biological fluids	Spectrophotometric Method A: Direct UV spectrophotometric measurement Method B: Reaction of drugs with N-bromosuccinimide (NBS)	Distilled water	Method A: Isoniazid: 2-42 Rifampicin: 0.822-65.38 Method B: Isoniazid: 0.1-3.4 Rifampicin: 0.5-15.9	Method A: Isoniazid: 264 nm Rifampicin: 474 nm Method B: Isoniazid: 572 nm Rifampicin: 572 nm	76
18	Isoniazid Rifampicin and Piperine in pharmaceutical dosage form	Spectrophotometric (Absorption correction method)	Methanol and Distilled water	Isoniazid: 12-34.5 Rifampicin: 8-23 Piperine: 0.4-1.15	Isoniazid: 262 nm Rifampicin: 477 nm Piperine: 338 nm	77
19	Isoniazid and Ethambutol HCl in pure form, pharmaceutical preparations and biological fluids	Spectrophotometric Method A (Reaction of Isoniazid with Iodine – starch solution Method B (Reaction of Isoniazid and Ethambutol hydrochloride with Hydroquinone solution)	Distilled water	Method A: Isoniazid: 1-6 Method B: Isoniazid: 2-100 Ethambutol HCl: 0.5-11.0	Method A: Isoniazid: 572 nm Method B: Isoniazid: 310 nm Ethambutol HCl: 218 nm	78
Rifampicin
1	Rifampicin in bulk, capsule & spiked human urine	Spectrophotometric	Method A: 0.1 M HCl Method B: 0.1 M H₃PO₄	1.5-30	Method A: 263 nm Method B: 259 nm	79
2	Rifampicin in bulk and capsule	Spectrophotometric	Methanol	5-13	337 nm	80
3	Rifampicin in a mixture of Isoniazid and Pyrazinamide	Spectrophotometric	Ethyl acetate	2.5-35.0	344 nm	81
4	Rifampicin pharmaceutical formulations	Visible Spectrophotometric	Buffer solution (pH=7.0)	5-50	510 nm	82
Rifampicin in combination with other drugs
5	Rifampicin and Isoniazid in combined dosage form	Spectrophotometric (Simultaneous Equation Method)	Ethanol	Rifampicin: 5-35 Isoniazid: 5-25	Rifampicin: 337 nm Isoniazid: 263 nm	83
6	Rifampicin and Piperine in combined capsule dosage form	Spectrophotometric (Second order derivative)	Methanol	Rifampicin: 10-60 Piperine: 2-20	ZCP for Rifampicin: 241 nm ZCP for Piperine: 341 nm	84
7	Rifampicin and Piperine in combined capsule dosage form	Spectrophotometric (Dual Wavelength)	Methanol	Rifampicin: 10-60 Piperine: 1-10	Rifampicin: 286 and 357 nm Piperine: 356 nm and 479 nm	85
8	Rifampicin and Piperine in combined capsule dosage form	Spectrophotometric (Q-absorption ratio)	Methanol	Rifampicin: 5-40 Piperine: 2-20	Iso-absorptive point: 387 nm Second wavelength: 337 nm	86
9	Rifampicin & Isoniazid in urine and pharmaceutical formulation	Spectrophotometric (Multivariate Visible)	Deionized water	Rifampicin: 8-57 Isoniazid: 1.5-7	Rifampicin: 449 nm Isoniazid: 455 nm	87
Ethambutol
1	Ethambutol in pure form and in pharmaceutical formulations	Spectrophotometric (Using triphenyl methane dyes viz., Bromocresol Green (BCG), Bromocresol Purple (BCP) and Bromophenol Blue (BPB))	Distilled Water	BCG: 2.0-25 BCP: 3.0-30 BPB: 4.0-40	BCG: 420 nm BCP: 419 nm BPB: 415 nm	88
2	Ethambutol in pure form & pharmaceutical formulations	Spectrophotometric (based on reaction of the drug with 2,4-dinitro-1-fluorobenzene under stipulated conditions)	Distilled Water	5-40	376 ± 1 nm	89
Pyrazinamide
1	Pyrazinamide in bulk and pharmaceutical dosage form	Spectrophotometric Method A: Area under curve Method B: Second order derivative	Water	Method A: 2-16 Method B: 2-16	Method A: 264-274 nm Method B: 270 nm	90
2	Pyrazinamide (PYN) and its impurity Pyrazine-2-carboxylic acid (PYA)	Spectrophotometric Method A: Third order derivative Method B: First order derivative	Methanol	PYN: 5-35 PYA: 5-30	Method A: PYN:276.2 nm PYA:274.6 nm Method B: PYN:225.8 nm PYA:245.2 nm	91
Pyrazinamide in combination with other drugs
3	Pyrazinamide (PYZ), Rifampicin (RIF) and Isoniazid (INH) in combined pharmaceutical dosage forms	Spectrophotometric (Second order derivative)	0.1N HCl	PYZ: 5-15 RIF: 6-12 INH: 6-18	PYZ:253.80 nm RIF:299.80 nm INH:302.40 nm	92
Rifabutin
1	Rifabutin in pharmaceutical formulations and in bulk drugs	Spectrophotometric Method A: Quantitative precipitation of RFB with iodine Method B: Quantitative precipitation of RFB with Tannic acid	Distilled water	Method A: 25-150 Method B: 10-60	Method A: 520 nm Method B: 460 nm	93
Rifapentine
1	Rifapentine in pure form and pharmaceutical formulations	Visible Spectrophotometric	0.1N HCl	5-50	478 nm	94
2	Rifapentine in bulk drug and tablets	Spectrophotometric (Area under curve)	Methanol	4-24	334 nm	95
Ethionamide
1	Ethionamide in bulk, tablet and nanoparticles	Spectrophotometric	Phosphate buffer (pH 7.4)	6-18	288 nm	96
2	Ethionamide in pharmaceuticals	Spectrophotometric	Methanol & water	5-25	288 nm	97
3	Ethionamide in pharmaceuticals	Spectrophotometric	Water	2.5-35	550 nm	98
4	Ethionamide in pharmaceuticals	Spectrophotometric Method A: Using Folin–Ciocalteu Method B: Using iron (III)-ferricyanide	0.1 M HCl & Water	Method A: 1-40 Method B: 0.2-4	Method A: 760 nm Method B: 760 nm	99
5	Ethionamide in pharmaceuticals	Spectrophotometric (Using two sulphonphthalein dyes) Method A: Using bromophenol blue Method B: Using bromothymol blue	Chloroform	Method A: 0.4-10 Method B: 0.5-14	Method A: 450 nm Method B: 450 nm	100
Cycloserine
1	Cycloserine in bulk & capsule dosage form	Spectrophotometric Method A:Area under curve Method B: First order derivative	0.01N HCl	5-25	Method A: 217 nm Method B: 217 nm	101
2	Cycloserine in pharmaceuticals	Spectrophotometric (Using Chloranil)	Borate buffer (pH 9)	2-8	348 nm	102
Terizidone
1	Terizidone in bulk and capsule dosage form	Spectrophotometric Method A:Area under curve Method B: First order derivative	0.1N NaOH	4-12	Method A: 273 nm Method B: 273 nm	103
p-Aminosalicylic Acid
1	p-Amino salicylic Acid in tablets	Spectrophotometric (Using derivatizing reagents) Method A: Using p-dimethylaminobenzaldehyde (DAB) Method B: Using p-dimethylaminocinnamaldehyde (DAC)	Ethanol Method A: 3M HCl-KCl buffer(pH 0.5) Method B: 5M HCl-KCl buffer (pH 0.5)	0.4-2.0	Method A: 460 nm Method B: 555 nm	104
Bedaquiline
1	Bedaquiline in bulk and pharmaceutical formulations	Spectrophotometric Method A: Zero order derivative Method B: Area under curve	Acetonitrile	15-75	285 nm	105

2. Spectrofluorimetric Methods:

TABLE 6: SPECTROFLUORIMETRIC METHODS

S. no.	Drug / Sample	Method	Solvent	Linearity Range	Wavelength of Detection [λex/λem]	Ref. no.
Isoniazid
1	Isoniazid (INH), Ethambutol (EMB), Pyrazinamide (PZA) and Rifampicin (RIF) in pure and pharmaceutical dosage forms	Spectrofluorimetric (Based on measuring the quenching effect of studied drugs on the fluorescence intensity of NBS-phenothiazine oxidation product (NBS-Phz))	Methanol	INH= 0.1-0.35 μg/ml EMB= 1-4 μg/ml PZA= 0.1-1 μg/ml RIF= 1-5	271/375 nm	106
Ethionamide
1	Ethionamide (ETN) & Carbocisteine (CBC) in their dosage forms	Spectrofluorimetric (Based on the reaction of drugs with roth's reagent (o-phthaldehyde) to get a highly fluorescent isoindole product)	Distilled water	ETN= 0.25-2.5 CBC= 0.05-0.9	ETN= 339/424 nm CBC= 329/431 nm	107
p-Aminosalicylic Acid
1	p-Aminosalicylic acid	Spectrofluorimetric	sodium acetate buffer (pH 4.0)	0.051-12 Mm	297/394 nm	108
2	p-Aminosalicylic acids (PAS) & p-A minobenzoic (PABA) in biological fluids	Spectrofluorimetric (Using terbium-sensitized Luminescence)	Water	PAS: 0-40 μmol/L PABA:0- 10 μmol/L	In alkaline solution= 324/546 nm In acidic solution= 292/546 nm	109

3. Chromatographic Methods:

A. Liquid Chromatographic Methods:

TABLE 7: LIQUID CHROMATOGRAPHIC METHODS

S. no.	Drug / Sample	Method	Column	Mobile phase	Flow Rate (ml/ min)	Detection	Ref. no.
Isoniazid
1	Isoniazid in human plasma	HPLC	VP-ODS C18 (250 mm x 4.6 mm, 5 μm)	Aquabidest: Acetonitrile (97:3, v/v)	1	UV 262 nm	110
2	Isoniazid in plasma, brain, liver and kidney samples and in solid lipid nanoparticles	HPLC	Waters, Symmetry Shield RP-18 (150 mm x 4.6 mm, 5 μm)	0.1 M phosphate buffer (pH 5 adjusted with ortho phosphoric acid) and methanol (50:50, v/v)	0.9	PDA 254 nm	111
3	Isoniazid in serum	HPLC	C18 (250 mm x 4.6 mm, 4 μm)	Acetonitrile,water, triethylamine & acetic acid (400:600:2:1, v/v/v/v)	1	UV 340 nm	112
4	Isoniazid in rat plasma	HPLC	C18 (150 mm x 4.6 mm, 5 μm)	Hexane sulphonate 20 mM (pH 2.47) & Methanol (65:35, v/v)	1	UV 265 nm	113
5	Isoniazid	HPLC	C18 (250 mm x 4.6 mm, 5 μm)	5.3% ethanol, 93.7% water, 1% acetic acid	1	UV 265 nm	114
6	Isoniazid in human plasma	HPLC	Pinnacle II C18 (150 mm x 4.6 mm, 5 μm)	0.05 M ammonium acetate buffer (pH 6): Acetonitrile (99:1, v/v)	1.2	UV 275 nm	115
Isoniazid in combination with other drugs
7	Isoniazid & Rifampicin in bulk and pharmaceutical formulations	RP-HPLC	XDB C18 (150 mm x 4.6 mm, 5 μm)	KH₂PO₄ buffer (pH 4.5): Methanol (60:40, v/v)	0.8	PDA 258 nm	116
8	Isoniazid & Rifampicin in nanoparticle drug formulations	RP-HPLC	Phenomenex Luna C18 (150 mm x 4.6 mm, 5 μm)	Methanol and water (10:90, v/v)	1	UV 268 nm	117
9	Isoniazid & Omeprazole determination in human serum	HPLC	Octasilil C8 (250 mm x 4.6 mm, 5 μm)	10 mM triethylamine pH 10.5: acetonitrile (67:33, v/v)	1	UV 260 nm	118
10	Isoniazid & Acetyl isoniazid in plasma	HPLC	ODS (150 mm × 3 mm, 3.5 μm)	20 mM 1-hexanesulfonic acid sodium salt solution (pH 3 adjusted with phosphoric acid) and acetonitrile in gradient elution program	0.4	UV 290 nm	119
11	Isoniazid and Acetyl Isoniazid in urine	HPLC	C8 (250 mm x 4.6 mm, 5 μm)	Water and methanol (80:20, v/v)	1.2	PDA 274 nm	120
12	Isoniazid (INH) & Ciprofloxacin Hydrochloride encapsulated in lipid polymeric hybrid nanoparticles	RP-HPLC	C18 (150 mm x 4.6 mm, 5 μm)	0.1% trifluoroacetic acid acetonitrile (70:30, v/v)	1	PDA 272 nm	121
13	Isoniazid and its related substances in Isoniazid and Ethambutol HCl tablet	HPLC	C18 (250 mm x 4.6 mm, 5 μm)	A potassium dihydrogen orthophosphate buffer of pH 6.9	1.5	PDA 254 nm	122
14	Isoniazid and Ethambutol in tablet dosage form	RP-HPLC	ODS C18 (250 mm x 4.6 mm, 5 μm)	0.05M Phosphate buffer (pH 4.6) and Acetonitrile (30:70, v/v)	1	PDA 255 nm	123
15	Isoniazid -pyridoxine HCl mixture	HPLC	ODS (250 mm x 4.6 mm, 5 μm)	Methanol: water (60:40, v/v)	2	293 nm	124
16	Isoniazid and Ethambutol in pharmaceuticals	HPLC	C18 Thermo Hypersil ODS, (250 mm x 5.4 mm, 4.5 μm)	Methanol: ammonium acetate buffer (pH-7.03) (50:50, v/v)	1.3	UV 276 nm	115
17	Isoniazid, Rifampicin in tablet dosage form	HPLC	Inertsil (250 mm x 4.6 mm, 5 μm)	Water (pH 4.5 adjusted with sodium dihydrogen phosphate): Acetonitrile (40:60, v/v)	1	UV 274 nm	125
18	Isoniazid, Ethambutol Hydrochloride & Rifampicin in tablet formulation	RP-HPLC	Prontosil C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile: 0.02M sodium dihydrogen phosphate buffer (pH 6.5 adjusted with orthophosphoric acid) (60:40, v/v)	1	UV 208 nm	126
19	Isoniazid, Thiacetazone and Pyridoxine HCl in tablet dosage form	RP-HPLC	Inertsil ODS Zodiac C18 (250 mm x 4.6 mm, 5 μm)	Ammonium Acetate: acetonitrile (30:70, v/v)	1	UV 254 nm	127
20	Isoniazid, Thiacetazone and Pyridoxine in tablet dosage form	RP-HPLC	Hypersil ODS C18 (150 mm x 4.6 mm, 5 μm)	Ammonium formate buffer: acetonitrile (60:40, v/v)	1	UV 254 nm	128
21	Isoniazid, Rifampicin & Piperine in pharmaceuticals	HPLC	E-Merck RP-18 (250 mmx 4.0 mm,5 μm)	Sol. A: Water + 0.1% acetic acid buffer, 2.5mM ammonium acetate Sol. B: Acetonitrile + 0.1% acetic acid buffer (10:90, v/v)	0.4	UV 263 nm	115
22	Isoniazid, Rifampicin, Piperine in pure & pharmaceutical dosage form	RP-HPLC	LC18 (250 mm x 4.6 mm, 5 μm)	0.01M Sodium dihydrogen orthophosphate, pH 6.5 and acetonitrile (40:60, v/v)	0.9	PDA 282 nm	77
Rifampicin
1	Rifampicin in complex pharmaceutical formulation and human serum	HPLC	Zorbax C18 (250 mm x 4.6 mm, 5 μm)	Methanol and water in gradient programme	1	UV 333.6 nm	129
2	Rifampicin in human plasma	HPLC	Phenomenex ODS C18 (150 mm x 4.6 mm, 5 μm)	Acetonitrile and 10mM potassium dihydrogen phosphate (pH adjusted to 3.2) (40:60, v/v)	1	UV 337 nm	130
3	Rifampicin in cerebrospinal fluid and plasma of the rabbit	HPLC	C8 (250 mm x 4.6 mm, 5 μm)	Acetonitrile: 10 mM phosphate buffer of pH 3.5 (48: 52, v/v)	1	215 nm	131
4	Rifampicin in human plasma	HPLC	Chromolith RP8 column (100 mm x 4.6 mm, 2 μm)	0.05 m acetate buffer pH 5.7: acetonitrile (35:65, v/v)	1	UV 335 nm	132
5	Rifampicin in dried bloods spots	HPLC	C-8 (Waters, Sunfire (250 mm x 4.6 mm, 5 μm)	50 mM ammonium acetate buffer pH 4.5, Acetonitrile and Methanol (40:30:30, v/v/v)	0.5	UV 261 nm	133
6	Rifampicin in bulk and pharmaceutical dosage form	RP-HPLC	C18 (250 x 4.6 mm, 3.5 μm)	Acetonitrile and water (80:20, v/v)	0.8	UV 237 nm	134
7	Rifampicin in plasma	RP-HPLC	C18 (250 mm x 4.0 mm, 4 μm)	phosphate buffer pH 7.4: methanol (75:25, v/v)	1.5	UV 475 nm	135
8	Rifampicin in bulk form and capsules	UPLC	Waters Acquity UPLC BEH C18 (100 mm x 2.1 mm, 1.7 μm)	Milli-Q water and acetonitrile (50:50, v/v)	0.4	UV 235 nm	136
9	Rifampicin in human serum	UPLC	BEH C18 (100 mm x 2.1 mm, 1.7 μm)	Acetonitrile & 0.05 M acetate buffer pH 4.0 (35:65, v/v)	0.5	UV 334 nm	137
10	Rifampicin in human Plasma, broncho-alveolar lavage fluid and alveolar cells	HPLC	Ultrasphere octyl (150 mm x 4.6 mm, 5 μm)	36% acetonitrile in water, 0.2% phosphoric acid, and 0.5% hydrogen peroxide adjusted to pH 4.5 with sodium hydroxide	1	Fluores-cence 380/490 nm	138
11	Rifampicin in serum	HPLC	Phenomenex Prodigy ODS (150 mm x 4.6 mm, 5 μm)	0.1mmol/L phosphate buffer pH 4.8: methanol (70:30, v/v)	1	335 nm	82
12	Rifampicin	RP-HPLC	ODS C18 (150 mm x 4.6 mm, 3.5 μm)	Potassium dihydrogen phosphate buffer (pH 3 adjusted with o-phosphoric acid) and acetonitrile (50:50, v/v)	1	PDA 238 nm	82
13	Rifampicin In tablet dosage form	RP-HPLC	C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile: 0.05M potassium phosphate buffer (38:62, v/v)	1	UV 335 nm	125
14	25-Desacetyl Rifampicin (25-DR) in human urine	HPLC	Agilent Eclipse XDB C18 (250 mm x 4.6 mm, 5 μm)	Methanol: 0.01 M sodium phosphate buffer pH 5.2 (65:35, v/v)	0.8	254 nm	139
Rifampicin in combination with other drugs
15	Rifampicin and 25-desacetyl-rifampicin in plasma	HPLC	C18 (250 x 4.6 mm, 5 μm)	Methanol and 0.058 M sodium nitrite solution (63:37, v/v)	4.7	UV 335 nm	140
16	Rifampicin and 25-O-Desacetyl Rifampicin in vitro metabolism	HPLC	A Phenomenex Luna C-18 (150 mm × 4.6 mm, 5 μm)	Water & methanol in gradient elution program	0.8	PDA 254 nm	141
17	Rifampicin and desacetyl rifampicin in plasma and urine	HPLC	Phenomenex Luna C18 (250 mm x 4.6 mm, 5 μm)	0.05 M phosphate buffer (pH 2.6): acetonitrile (55:45, v/v)	1.2	PDA 254 nm	142
18	Rifampicin and related compounds in pharmaceuticals	HPLC	C18 monolithic (100 mm x 4.6 mm, 5 μm)	Methanol, acetonitrile, 0.075 M monopotassium phosphate & 1.0 M citric acid (28:30:38:4, v/v)	2	UV 254 nm	143
19	Rifampicin & Clindamycin phosphate in skin permeation studies	HPLC	C18 (150 mm x 4.6 mm, 5 μm)	0.01 M phosphoric acid and methanol in gradient elution program	1	UV 238 nm 200 nm	144
20	Rifampicin &Daptomycin in rabbit plasma	UPLC	Acquity BEH C18 (100 mm x 2.1 mm, 1.7 μm)	Methanol and 0.1% aqueous TFA in gradient elution program	1	UV	145
21	Rifampicin and a flavonoid glycoside	RP-HPLC	RP-18 (250 mm × 4.6 mm, 5 μm)	Acetonitrile & 50 mM phosphate buffer (pH 5.0) (60:40, v/v)	0.8	DAD 340 nm	146
22	Rifampicin and Sulbactam in mouse plasma	HPLC	RP-18 (125 mm × 4.0 mm, 5 μm)	50 mM potassium dihydrogen phosphate solution (pH 4.5) and acetonitrile in gradient elution program	1	DAD 230 nm	147
23	Rifampicin and Piperine in pharmaceutical dosage form	RP-HPLC	C18 (250 mm x 4.6 mm, 5 μm)	Potassium dihydrogen orthophosphate pH 6.5 and acetonitrile (30:70, v/v)	1	PDA 341 nm	148
24	Rifampicin and Ofloxacin in synthetic mixture	HPLC	Kinetex C18, Phenomenex (250 mm x 4.6 mm, 5 μm)	0.03M Potassium dihydrogen phosphate buffer pH 3.0: acetonitrile (55:45, v/v)	0.8	PDA 230 nm	149
25	Rifampicin and Isoniazid in human plasma	HPLC	1.Luna C18 (250 mm x 4.6 mm, 5 μm) 2.Luna C8 (250 mm x 4.6 mm, 5 μm)	1.Methanol:0.02M Potassium phosphate buffer pH 7.0 (75:25, v/v) 2.Methanol: water: perchloric acid: tetrabutylammonium hydroxide solution (20:80:0.05:0.05, v/v/v/v)	0.5 & 1	PDA 339 nm 273 nm	150
26	Rifampicin and Isoniazid in pharmaceutical formulations	RP-HPLC	Kromasil C18 (250 mm x 4.6 nm, 5 μm)	Methanol, acetonitrile and water (60:20:20, v/v/v)	1	UV 254 nm	151
27	Rifampicin & Hydro-chlorothiazide	HPLC	Phenomenex ODS 2 C18 (150 mm x 4.6 mm, 5 μm)	Acetonitrile and 10mM KH₂PO₄ (pH 3.2) (40:60, v/v)	1	337 nm	82
28	Rifampicin and Isoniazid	HPLC	ODS (250 mm x 4.6 nm, 5 μm)	Methanol:0.02M disodium hydrogen orthophosphate (75:25, v/v)	1	254 nm	82
29	Rifampicin & isoniazid	HPLC	C18 (250 mm x 4.6 mm, 5 μm)	0.05M sodium dihydrogen phosphate (pH 3.1) and acetonitrile (20:80, v/v)	0.6	254 nm	82
Ethambutol
1	Ethambutol in human plasma	HPLC	CN (150 mm x 4.6 mm, 5 μm)	Milli-Q water and methanol (85:15, v/v)	1.5	PDA 267 nm	152
2	Ethambutol in serum	LC	Waters C18 (150mm x 4.6 mm, 5 μm)	Aqueous 72% (v/v) acetonitrile	1	Fluore-scence 345/475 nm	153
3	Ethambutol in rat plasma	UPLC	BEH RP 18 (50 mm x 2.1 mm, 1.7 μm)	Methanol and water (70: 30, v/v)	0.1	PDA 205 nm	154
4	Ethambutol in pharmaceutical dosage form	RP-HPLC	C18, (50 mm x 4.6 mm, 5 μm)	Dichloromethane: methanol: formic acid (70:30:0.1, v/v/v)	1	225 nm	125
Ethambutol in combination with other drugs
5	Ethambutol Hydrochloride and Isoniazid in fixed dose formulation	RP-HPLC	C18 Thermo Hypersil ODS (250 mm x 5.4mm, 4.5 μm)	Methanol: ammonium acetate buffer (pH-7.03)(50:50, v/v)	1.3	PDA 276 nm	155
Pyrazinamide
1	Pyrazinamide in human plasma	HPLC	Supelco LC-18 (150 mm x 4.6 mm, 5 μm)	0.02 M phosphate buffer (pH 7.4) & methanol (96.8:3.2, v/v)	1.5	UV 268 nm	156
2	Pyrazinamide in human plasma	HPLC	Phenomenex ODS C18 (150 mm x 4.6 mm, 5 μm)	Methanol: potassium dihydrogen phosphate buffer (pH 7.4) (15:85, v/v)	1	UV 268 nm	157
3	Pyrazinamide in human plasma	HPLC	ODS C18 (250 mm x 4.6 mm, 5 μm)	Aquabidest: Acetonitrile (97:3, v/v)	1	UV 262 nm	158
4	Pyrazinamide in human plasma, bronchoalveolar lavage, and alveolar cells	HPLC	ODS C18 (250 mm x 4.6 mm, 5 μm)	2.0% acetonitrile in 0.02M KH2PO4, adjusted to pH 2.6 with phosphoric acid	1	UV 268 nm	159
5	Pyrazinamide in bulk and pharmaceutical dosage forms	HPLC	Hypersil C8 (250 mm x 4.6 mm, 3.5 μm)	Phosphate buffer (pH 4.4): methanol (80:20, v/v)	1	UV 269 nm	160
6	Pyrazinamide in tablet dosage form	RP-HPLC	C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile and 15mM potassium dihydrogen (pH 4.0 ± 0.1 adjusted with o-phosphoric acid) (11:89, v/v)	1	235 nm	125
7	Pyrazinamide in bulk and formulation	UHPLC	C18 (25 mm x 4.6 mm, 1.7 μm)	Phosphate buffer: acetonitrile (900:100, v/v)	1	PDA 270 nm	161
8	Pyrazinamide in pharmaceutical formulation	Micellar LC	SPHER-100 C18 (250 mm x 4.6 mm, 5 μm)	0.15M sodium dodecyl sulphate and 1% butanol (v/v) buffered at pH3 in gradient elution program	1	UV 269 nm	162
Pyrazinamide in combination with other drugs
9	Pyrazinamide and Rifampicin in serum	HPLC	C8 (250 mm x 4.6 mm, 3.5 μm)	Acetonitrile in 10 mM potassium dihydrogen phosphate (pH 3.5) in gradient elution program	1.5	215 nm	163
10	Pyrazinamide and Isoniazid in plasma	HPLC	Zorbax Eclipse Plus C18 (150 mm x 4.6 mm, 5 μm)	Acetonitrile and 20 mM 1-hexane sulfonic acid sodium salt (pH 2.7 adjusted with 10 % ortho-phosphoric acid) in gradient elution program	1	UV 269 nm & 340 nm	164
11	Pyrazinamide and Isoniazid in plasma	HPLC	Wakosil C18 HG (250 mm x 4.6 mm, 5 μm)	Acetonitrile and 0.05M ammonium acetate solution (1:99, v/v)	1.2	UV 275 nm	165
12	Pyrazinamide and Isoniazid in plasma	HPLC	C8 (250 mm x 4.6 mm, 5 μm)	Water: methanol (80:20, v/v)	1.5	UV 267 nm	166
13	Pyrazinamide and Isoniazid in synthetic mixture	RP-HPLC	Inertsil-ODS C18 (250 mm x 4.6 mm, 5 μm)	Methanol: buffer (pH 4 adjusted with triethylamine) (55:45, v/v)	1	UV 267 nm	167
14	Pyrazinamide & Ethionamide from their porous microparticles	HPLC Ion-Pair	Phenomenex Luna C18 (250 mm x 4.6 mm, 5 μm)	0.01% TFA in water and ACN/MeOH (50:50, v/v) in gradient elution program	1.5	UV 280 nm	168
15	Pyrazinamide, Rifampicin and Isoniazid in combined dosage forms	HPLC	YMC-ODS (150 mm x 4.6 mm, 5 μm)	Water, monobasic potassium dihydrogen orthophosphate and acetonitrile (900:60:40, v/v/v)	1.5	UV 254 nm	169
16	Pyrazinamide, Rifampicin and Isoniazid in pharmaceutical preparations	HPLC	Phenomenex C18 (250 mm x 4.6 mm, 5 μm)	Methanol, water, isopropanol, acetonitrile & 1mM sodium acetate (51:42:3:2:2, v/v/v/v/v)	1.7	UV 333 nm	170
17	Pyrazinamide, Rifampicin and Isoniazid in 0.1M HCl dissolution Medium and Simulated Gastric Fluid	HPLC	Suspelcosil LC18 (250 mm x 4.6 mm, 5 μm)	Methanol and 0.01M sodium dihydrogen orthophosphate buffer containing 0.05% tetramethyl-ammonium chloride (pH 3.5 adjusted with dil. orthophosphoric acid) in gradient elution program	1	UV 254 nm	171
18	Pyrazinamide, Rifampicin and Isoniazid in fixed dose combination	HPLC	μ-bondapak C18 (250-mm x 4.6 mm, 10 μm)	ACN:0.0002M tBAH (42.5:57.5, v/v)	1	UV 260 nm	172
19	Pyrazinamide (PYZ) Rifampicin (RIF) & Isoniazid (INH) in plasma	HPLC	For PYZ &INH Spherisorb C8 (150 mm x 4.6 mm, 5 μm) For RIF Spherisorb C8 (250 mm x 4.6 mm, 5 μm)	For PYZ &INH: 3% acetonitrile in 0.06% TFA For RIF: 80% acetonitrile in 0.1% trifluoroacetic acid	2 & 1.5	For PYZ &INH UV 254 nm For RIF UV 270 nm	173
20	Pyrazinamide, Rifampicin and Isoniazid in human plasma	RP-HPLC	Phenomenex ODS C18 (250 mm x 4.0 mm, 5 μm)	Acetonitrile, methanol andwater (pH5.2) (30:5:65, v/v/v)	1	UV 242 nm	174
21	Pyrazinamide, Rifampicin and Isoniazid in solid lipid nanoparticles	RP-HPLC	ODS C18 (250 mm x 4.0 mm, 5 μm)	A.OPA buffer (pH 6.8 ± 0.02 with dil. NaOH): acetonitrile (96:4, v/v) B. OPA buffer (pH 6.8 ± 0.02 with dil.NaOH): acetonitrile (45:55, v/v) Mobile phase A & B in gradient elution program	1.5	PDA 238 nm	175
22	Pyrazinamide, Isoniazid and Indomethacin in pharmaceutical preparation	HPLC	YMC-ODS (150 mm x 4.6 mm, 5 μm)	Water, methanol & tetrahydrofuran (59:39:2, v/v/v)	2	UV 328 nm	176
23	Pyrazinamide, Rifampicin and Isoniazid in tablet dosage form	RP-HPLC	Hypersil C18 (250 mm x 4.6 mm, 5 μm)	0.05 M potassium phosphate buffer (pH 6.0): Methanol (40:60, v/v)	1	UV 254 nm	125
24	Pyrazinamide (PZA), Rifampicin (RIF) Isoniazid (INH) & Acetyl-isoniazid (AcINH) in Human Plasma	HPLC	Synergi Max-RP C12 (250 mm x 4.6 mm, 4 μm)	Methanol, acetonitrile and buffer of 20 mM 1-heptanesulfonic acid sodium (pH2.5 adjusted with H₃PO₄) in gradient elution program	0.8, 1.2, 1.5	DAD PZA= 268 nm AcINH= 265 nm INH= 264 nm RIF= 341 nm	177
25	Pyrazinamide, Rifampicin, Isoniazid & Ethambutol HCl in fixed dose combination tablet	HPLC	Waters Symmetry C8 (250 mm x 4.6 mm, 5 μm)	Acetonitrile and 20 mM phosphate buffer (pH 6.8)containing triethylamine in gradient elution program	1.5	UV 210 nm	178
26	Pyrazinamide, Rifampicin, Isoniazid & Ethambutol HCl in fixed dose combination tablet	RP-HPLC	Waters Xterra RP18 (250 mm x 4.6 mm, 5 μm)	phosphate buffer (pH 6.8), 8% acetonitrile and acetate buffer (pH 4.7) in gradient elution program	1	UV 260 nm	125
27	Pyrazinamide (PYZ), Rifampicin (RIF), Isoniazid (INH), and Ethambutol hydrochloride (EMB) in fixed dose combination tablet	HPLC	Purospher STAR RP18e (250 mm x 4.6 mm, 5 μm)	20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile in gradient elution program	1.5	DAD PYR, RIF & INH = 238 nm EMB = 210 nm	179
28	Pyrazinamide (PYZ), Rifampicin (RIF), Isoniazid (INH) and Ethambutol hydrochloride (EMB) in fixed dose combination tablet	HPLC	Acclaim Polar Advantage II (150 mm x 4.6 mm, 3 μm)	A: 8% Acetonitrile in 20 mM NaH₂PO₄ (plus 1.5 mL TEA per liter), pH 6.8 B: 50% Acetonitrile in 20 mM NaH₂PO₄ (plus 1.5 mL TEA per liter), pH 6.8	1	UV Channel-1 200 nm & 337 nm Channel-2 238nm	180
29	Pyrazinamide (PYZ), Rifampicin (RIF), Isoniazid (INH), and Ethambutol hydrochloride (EMB) in fixed dose combination tablet	UHPLC	Acclaim Polar Advantage II (100 mm x 2.1 mm, 2.2 μm)	A: 4% Acetonitrile in 20mM NaH₂PO₄ (plus 1.5 mL TEA per liter), pH 6.8 B: 50% Acetonitrile in 20 mM NaH₂PO₄ (plus 1.5 mL TEA per liter), pH 6.8	1	UV Channel-1 200 nm & 337 nm Channel-2 238nm	180
30	Pyrazinamide (PYZ), Rifampicin (RIF), Isoniazid (INH), and Ethambutol hydrochloride (EMB) in fixed dose combination tablet	UHPLC	Waters Acquity BEH C18 (50 mm x 2.1 mm, 1.7 μm)	Triethylamine in phosphate buffer pH 6.8 and acetonitrile (95:5, v/v)	0.4	UV PYR, RIF & INH = 238 nm EMB = 210 nm	181
31	Pyrazinamide, Rifampicin, Isoniazid & Ethambutol HCl in fixed dose combination tablet	UPLC	X bridge C18 (50 mm x 1.7 mm, 3 μm)	Solution-A: Triethylamine and potassium dihydrogen ortho- Phosphate buffer (pH 7.5 adjusted with ortho phosphoric acid) Solution-B: Mixture of methanol and acetonitrile (85:15, v/v) -Mixture of solution- A and solution-B (90:10, v/v)	0.5	PDA 290 nm	182
32	Pyrazinamide, Rifampicin, Isoniazid & Pyridoxine HCl in pharmaceutical formulation	RP-HPLC	Phenomenex Luna C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile and 15 mmol/L potassium dihydrogen phosphate buffer (pH 4.0 ± 0.1 adjusted by orthophosphoric acid) in gradient elution program	1	PDA 235 nm	183
Rifabutin
1	Rifabutin in human plasma	HPLC	C18 (250 mm x 4.6 mm, 5 μm	50mM phosphate buffer, (pH 4.2 adjusted with 1N HCl) & acetonitrile (53:47, v/v)	1.2	UV 265 nm	184
2	Rifabutin	HPLC	C18 (250 mm x 4.6 mm, 5 μm	Acetonitrile + methanol (1:1): water (75:25, v/v)	1	UV 242 nm	185
3	Rifabutin in bulk dosage form	RP-HPLC	Phenomenex C8 Luna (250 mm x 4.6 mm, 5 μm)	Methanol and water (75:25 v/v)	1	UV 240 nm	186
4	Rifabutin in bulk drugs and pharmaceutical dosage form	Stability LC	Ace5-C18 (250 mm x 4.6 mm, 5 μm)	50 mM ammonium acetate (pH 4 adjusted by acetic acid) and acetonitrile (50:50, v/v)	1	UV 275 nm	187
5	Rifabutin in human plasma	HPLC	Zorbax C8 (250 mm x 4.6 mm, 5 μm)	0.05 M potassium dihydrogen phosphate0.05 M sodium acetate at pH 4.0: acetonitrile (53:47, v/v)	1	UV 275 nm	188
Rifabutin in combination with other drugs
6	Rifabutin and 25-O-desacetyl rifabutin in human plasma and urine	HPLC	ODS (250 mm x 4.6 mm, 5 μm)	Acetonitrile, 0.05 M potassium phosphate (pH 4.2) & Triethylamine (38:61.5:0.5, v/v/v)	1	UV 275 nm	189
Rifapentine
1	Rifapentine in bulk and pharmaceutical dosage form	RP-HPLC	Inertsil C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile and 0.01M potassium dihydrogen phosphate buffer, pH (6.0), (80:20, v/v)	0.8	UV/Visible 478 nm	190
2	Rifapentine	HPLC Impurity profile	BDS-Hypersil C18 (250 mm x 4.6 mm, 5 μm)	A mixture of 0.025M sodium dihydrogen orthophosphate buffer (pH 7.7 adjusted with dil. NaOH) and ACN (90:10, v/v) for mobile phase A (30:70, v/v) for mobile phase B. Use Mobile phase A & B in gradient elution program	1	PDA 254 nm	191
Ethionamide
1	Ethionamide in human plasma	HPLC	CN (150 mm x 4.6 mm, 5 μm)	Milli-Q water and methanol (85:15, v/v)	1.5	PDA 267 nm	192
2	Ethionamide in Serum	HPLC	Hypersil ODS C18 (250 mm x 4.6 mm, 5 μm)	0.02 M disodium hydrogen phosphate buffer: acetonitrile (75:25, v/v)	1.5	UV 291 nm	193
3	Ethionamide in dosage form	RP-HPLC	Hypersil BDS C18 (150 mm x 6 mm, 3 μm)	Acetonitrile: water (30:70, v/v)	1	UV 287 nm	194
4	Ethionamide in raw material & pharmaceutical dosage forms	Stability HPLC	ODS C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile: 0.05% trifluoroacetic acid solution (30:70, v/v)	0.8	UV 270 nm	195
5	Ethionamide in spiked human plasma	RP-HPLC	C18 (250 mm x 4.6 mm, 5 μm)	Methanol: water (40:60, v/v)	1	UV 275 nm	196
6	Ethionamide in pharmaceutical dosage forms	RP-HPLC	Grace C18 (250 mm x 4.6 mm, 5 μm)	Methanol: 0.1% Ortho Phosphoric acid (20:80, v/v)	0.7	UV 288 nm	97
Ethionamide in combination with other drugs
7	Ethionamide, Pyridoxine, and Moxifloxacin in fixed dose combination tablets	Stability RP-HPLC	Hibar RP 18 (150 mm x 4.6 mm, 5 μm)	0.03M sodium citrate buffer (pH 5.0 adjusted with glacial acetic acid) and methanol in gradient elution program	1	UV 320 nm	197
Cycloserine
1	Cycloserine in human plasma	HPLC	C18 (250 mm x 4.6 mm, 5 μm)	0.1% formic acid solution and a mixture of methanol and acetonitrile (1:1) (85:15, v/v)	1	Fluore-scence 381/450 nm	198
2	D-Cycloserine & related substance	LC	Hypersil BDS C18 (250 mm x 4.6 mm, 5 μm)	Acetonitrile, 20mM sodium octane sulphonate, 0.2M potassium dihydrogen phosphate buffer pH 2.8 & water in gradient elution program	1	UV 219 nm	199
3	D-Cycloserine drug substance	RP-HPLC	Agilent Zorbax SB phenyl (250 mm x 4.6 mm, 5 μm	20mM Na₂HPO₄ (pH 7 adjusted with ortho-phosphoric acid) and acetonitrile (95:5, v/v)	1	UV 335 nm	200
Terizidone
1	Terizidone	Stability RP-HPLC	HiQSil C8 (250 mm x 4.6 mm, 5 μm)	Ammonium acetate buffer (pH 3 adjusted with glacial acetic acid) and methanol (60:40, v/v)	1	PDA 264 nm	201
2	Terizidone in plasma	HPLC	HS C18 (150 mm x 4.6 mm, 5 μm)	Acetonitrile and water both containing 0.1% formic acid in gradient elution program	1	UV 264 nm	202
p-Aminosalicylic Acid
1	p-Aminoslicylic acid and its metabolite in plasma, cerebrospinal fluid and brain tissues	HPLC	C18 (250 mm x 4.6 mm, 5 μm)	17.5 mM potassium phosphate buffer (equal molar concentration of both monobasic and dibasic potassium salts with a pH of 3.5 adjusted by phosphoric acid) and methanol in gradient elution program	1	Fluorescence 337/432 nm	203
2	p-Aminosalicylic acid (PAS) and its degradation product m-aminophenol (MAP) in pellets	Ion-pair HPLC	LiChrospherRP 18 (125 mm x 4 mm, 5μm)	20 mM phosphate buffer, 20 mM tetrabutylammonium hydrogen sulphate & methanol (16%, v/v) (pH 6.8) gradient elution program	1	UV 233 nm	204
Bedaquiline
1	Bedaquiline	RP-HPLC	Chiralcel OJ-3R (cellulose tris-[4-methylphenyl]benzoate, 150 mm x 4.6 mm, 3 μm)	10 mM buffer of triethylamine/phosphoric acid pH 7.0 and acetonitrile (40:60, v/v)	0.1-1.4	UV 227 nm	205
Bedaquiline in combination with other drugs
2	Bedaquiline (BED), Moxifloxacin (MOX) & Pyrazinamide (PYZ) in pharmaceutical powder formulation for inhalation	RP-HPLC	Luna C18 (150 mm x 4.6 mm, 5 μm)	Methanol and triethylamine phosphate buffer (pH 2.5) in gradient elution program	1.2	PDA BED= 225 nm MOX= 296 nm PYZ= 269 nm	206
Pretomanid
1	Pretomanid (PA-824), Moxifloxacin (MOX) and Pyrazinamide (PYZ) in an inhaler	HPLC	Luna C18 (150 mm x 4.6 mm, 5 μm)	Methanol and trimethylamine phosphate buffer (pH 2.5) in gradient elution program	1	PDA PRM= 330 nm MOX= 296 nm PYZ= 269 nm	207

B. Thin Layer Chromatographic Methods:

TABLE 8: THIN LAYER CHROMATOGRAPHIC METHODS

S. no.	Drug / Sample	Method	Stationary Phase	Mobile phase	Retention factor (R_f)	Detection	Ref. no.
Isoniazid in combination with other drugs
1	Isoniazid (INH) and Acetyl isoniazid (AcINH) in serum	HPTLC	Silica gel 60	Ethyl Acetate: methanol (70:30, v/v)	INH= 0.35 AcINH= 0.5	254 nm	208
2	Isoniazid (INH) and Rifampicin (RIF) in bulk drugs and formulations	Stability indicating HPTLC	Silica gel 60 F254	n-hexane, 2propanol, acetone, ammonia, formic acid, (3:3.8:2.8:0.3:0.1, v/v/v/v/v)	INH = 0.59±0.02 RIF= 0.73±0.04,	254 nm	209
3	Isoniazid (INH) and Rifabutin (RFB) in pharmaceutical formulation	Stability indicating HPTLC	Silica gel 60 F254	Dichloromethane, acetone, methanol (20:7:2, v/v/v)	INH= 0.48±0.01 RFB= 0.84±0.01	INH= 262 nm RFB= 504 nm	210
4	Isoniazid (INH), Pyridoxine hydrochloride (PYR) and Rifampicin (RIF) in combined tablet dosage form	HPTLC	Precoated silica gel 60 G F254 aluminium sheet	Ethyl acetate: methanol: acetone: acetic acid (5.5: 2.0: 2.0: 0.5, v/v/v/v)	INH= 0.47±0.01 PYR= 0.75±0.01 RIF= 0.27±0.01	254 nm	211
Rifampicin in combination with other drugs
1	Rifampicin (RIF) and Isoniazid (INH) in rat plasma	HPTLC	Silica gel 60 F254	Chloroform: methanol (9:1, v/v)	RIF= 0.27±0.01 INH= 0.47±0.01	RIF= 475 nm INH= 280 nm	212
Pyrazinamide in combination with other drugs
1	Pyrazinamide (PYN) and its impurity Pyrazine-2-carboxylic acid (PYA)	HPTLC	Silica gel 60 F254	Methylenechloride: methanol: ammonia solution (7:3:0.1, v/v/v)	PYN= 0.86 PYA= 0.16	275 nm	91
2	Pyrazinamide (PYZ), Rifampicin (RIF) & Isoniazid (INH)in a fixed dosagecombination tablet	HPTLC	Silica gel 60 F254 plates	Acetate, acetone, methanol, glacial acetic acid with the ratio of (18:5:5:2, v/v/v/v)	PYZ=0.74 RIF=0.25, INH=0.44	277 nm	213
Terizidone
1	Terizidone in pharmaceutical dosage form	HPTLC	Silica gel 60 F254	Toluene: n-butanol (9:1, v/v)	0.60±0.03	268 nm	214

4. Gas Chromatography:

TABLE 9: GAS CHROMATOGRAPHY

S. no.

Drug / Sample

Description

Ref. no.

Isoniazid in combination with other drugs

Isoniazid (INH) and Hydrazine (HZ) in

pharmaceutical

preparations & blood

Capillary column gas chromatography after precolumn derivatization with trifluoroacetylacetone (FAA). Phenylhydrazine (PHZ) when present together with INH and HZ also separated completely from the column HP‑5 (30 mm x 0.32 mm) connected with flame ionization detection (FID). The solvent was evaporated under nitrogen gas and re-dissolved in 0.2 mL of methanol. The total run time was 7 min and nitrogen flow rate was 1mL/min. The linear calibration ranges for INH and HZ were determined to be 2.5-25 μg/mL and 2.5-21.2 μg/mL respectively, the detection limits were obtained at 62.5 pg reaching to the detector.

215

5. Micellar Electrokinetic Capillary Chromatography:

Table 10: MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY

S. no.

Drug / Sample

Method

Stationary Phase

Mobile

phase

Flow Rate

(ml/ min)

Detection

Ref. no.

Isoniazid in combination with other drugs

Isoniazid

(INH), Pyrazinamide (PYR) and Rifampicin (RIF) in pharmaceutical

products

MEKC

Nova-Pak C18 (150 mm x 3.9 mm, 4 μm)

Methanol in 20mM phosphate buffer & methanol in gradient elution program

254 nm

216

6. Electrochemical Methods:

Table 11: ELECTROCHEMICAL METHODS

S. no.	Drug/ Sample	Method	Electrode		Linearity Range	LOD	Ref. no.
S. no.	Drug/ Sample	Method	Working Electrode	Reference Electrode	Linearity Range	LOD	Ref. no.
Isoniazid
1	Isoniazid	Voltammetry (Differential Pulse Voltammetry)	Mercury film silver-based electrode (Hg (Ag) FE)	Ag/AgCl/ KCl	5-500 nM	4.1 nM	217
2	Isoniazid in urine	Amperometry	Glassy carbon electrode	Ag/AgCl/ KCl	0.05-783.1 μM	0.01 μM	218
3	Isoniazid	Voltammetry (Using poly (3,4-ethylenedioxythiophene)-modified gold electrode)	Crystalline Au (111)	Ag/AgCl/3M NaCl	0.05-2 μM	0.014 μM	219
4	Isoniazid in tablets	Amperometric	Glassy carbon electrode	Ag/AgCl	2.5 x 10^-8-1.0 x 10^-3M	4.1 x 10^-9M	220
Isoniazid in combination with other drugs
5	Isoniazid (INH) and Rifampicin (RIF) in pharmaceutical formulations	Voltammetry (Differential Pulse Voltammetry)	Hanging mercury drop electrode (HMDE)	Ag/AgCl/ KCl	INH= 0.25-1.25 mg/L RIF= 0.40-2.00 mg/L	INH= 0.05 mg/L RIF= 0.07 mg/L	221
6	Isoniazid (INH) & Acetaminophen (AAP) in human fluids	Voltammetry	Bismuth oxide modified screen- printed electrode	Ag/AgCl	INH= 5-1760 μM AAP= 0.5-1250 μM	INH= 1.85 μM AAP= 30 nM	222
Pyrazinamide
1	Pyrazinamide	Voltammetry	Screen-printed carbon electrode (SPCE)	Ag/AgCl	9.0 x 10^-7- 1.0 x 10^-4 mol /L	5.7 x 10^-7 mol/L	223
Ethionamide
1	Ethionamide in Pharmaceutical Formulations	Voltammetry	Boron-doped diamond electrode	Ag/AgCl	1.00-80.0 μmol/ L	0.294 μmol/L	224
Ethionamide in combination with other drugs
2	Ethionamide (ETH) and Pyrazinamide (PYZ)	Voltammetry	Glassy carbon electrode	Ag/AgCl	ETH= 2.38-248.0 µmol /L PYZ= 0.476-51.2 µmol/L	ETH= 0.531 µmol /L PYZ= 0.113 µmol /L	225
Cycloserine
1	D-Cycloserine in pharmaceutical and human biological samples	Voltammetry	Gold electrode	Ag/AgCl/ KCl	0.1-1.1 μM	3.3 x 10^-8M	226
2	D-Cycloserine in pharmaceutical products	Voltammetry (Stair Case (SCV) and Square Wave (SWV)	Graphene paste electrode	Ag/AgCl	SCV= 1.0×10^-8 - 1.5×10^-7 M SWV= 1.0×10^-8 - 1.1×10^-7 M	SCV= 2.80 nM SWV= 3.70 nM	227

7. Titrimetric Methods:

TABLE 11: TITRIMETRIC METHODS

S. no.	Drug / Sample	Description	Ref. no.
Isoniazid
1	Isoniazid	N-bromophthalimide used as a titrant. The end-point is determined either directly using methyl red or amaranth as indicator, or by a back-titration method in which a known excess of N-bromophthalimide solution is added to isoniazid solution and then the residual unreacted reagent is determined iodometrically.	70
2	Isoniazid	Titration of isoniazid with 0.02 M acetous perchloric acid in glacial acetic acid using crystal violet as an indicator. The method is applicable over the range of 1.5-15 mg isoniazid	228
Ethambutol
1	Ethambutol	0.2 gm of pure powder or 0.2 gm equivalent of ethambutol hydrochloride powder (in case of tablet) was taken in a 250 ml separating flask and 10 ml of 2N sodium hydroxide was added to the powder and was shaken thoroughly. The solution was titrated with 0.1 N perchloric acid solution using 0.5 ml of 0.1% methyl red indicator (end point pink violet).	229
Ethionamide
1	Ethionamide in pharmaceuticals	A 10 mL aliquot of standard Ethionamide solution containing 1.5-15 mg of Ethionamide was measured accurately and transferred into a 100 mL titration flask, 5 mL of 2M H2SO4 was added and titrated immediately against 0.01M KMnO4 to a first appearance of pink color.	98
2	Ethionamide in pharmaceuticals	A 10 mL aliquot of the pure Ethionamide solution containing 2-10 mg of drug was placed in a 100 mL titration flask. 25 mL of saturated sodium bicarbonate was added followed by 1 mL starch indicator. The content was titrated with standard iodine solution to a blue end point.	98
3	Ethionamide in pharmaceuticals	A 10 mL aliquot of the drug solution containing 2-9 mg of Ethionamide was measured accurately and transferred into a 100 mL titration flask followed by the addition of 5 mL of 2M HCl. Two drops of methyl orange indicator were added and content titrated vs 5 mM bromate-bromide mixture to a colorless end point.	98

8. Other Methods:

A. Liquid Chromatography/Mass Spectrometry Methods:

TABLE 12: LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY METHODS

S. no.	Drug / Sample	Method	Stationary Phase	Mobile phase	Flow Rate (ml/ min)	Detection/ m/z	Ref. no.
Isoniazid
1	Isoniazid in dog plasma	LC–MS	C18	0.1% formic acid:acetonitrile (91:9, v/v)	1	Mass spectrometric 138	230
2	Isoniazid levels in small hair samples	LC/MS-MS	Phenomenex Synergi Polar-RP (100 mm x 2.1 mm, 2.5 μm)	Water with 0.2% (v/v) formic acid	0.4	Mass spectrometric 79.0	231
3	Isoniazid	SFC-MS/MS	Inertsil ODS C18 (150 mm x 4.6 mm, 5 μm)	Dichloromethane: methanol: ethyl acetate: formic acid(70:30:0.5:0.1, v/v/v/v) (15%) and supercritical CO₂ (85%)	-	Mass spectrometric138	232
Isoniazid in combination with other drugs
4	Isoniazid (INH) and Ethambutol (EMB) in dried blood spots	LC/MS-MS	Kromasil C18 (150 mm x 4.6 mm, 5 μm)	0.1% formic acid in water and methanol (35:65, v/v)	0.8	Mass spectrometric INH 138.10 →121.10 EMB 205.20 → 116.10	233
5	Isoniazid (INH) and Ethambutol (EMB) in human plasma	LC/MS-MS	Atlantis Waters C18 (150 mm x 2.1 mm, 3 μm)	Methanol: water: formicacid (10:90:0.3, v/v/v)	0.20	Mass spectrometric INH= 205 → 116 EMB= 130 → 60	234
Rifampicin
1	Rifampicin in human plasma	LC/MS-MS	Kinetex C18 (50 mm x 2.1 mm, 2.6 ?m)	0.1% formic acid in water and acetonitrile in gradient elution program	0.5-0.9	Mas spectrometric 823.4 → 107.1 and 823.4 → 163.1	235
2	Rifampicin in human plasma and cerebrospinal fluid	LC/MS-MS	Hypersil–Hypurity C18 (150 mm x 2.1 mm, 5 μm)	ACN containing formic acid (0.05%, v/v) and 15 mMammonium formate buffer (pH 5) in gradient elution program	0.35	Mass spectrometric 823.4 → 791.4	236
3	Rifampicin in plasma	LC/MS-MS	BDS Hypersil Gold C18 (50 mm x 3 mm)	Methanol: 2 mM ammonium acetate (80:20, v/v)	0.2	Mass spectrometric	237
4	Rifampicin (RIF) in rat plasma	UPLC-MS/MS	BEH C18 (50 mm x 2.1 mm, 1.7 μm)	Acetonitrile and water (both containing 0.1 % formic acid) in gradient elution program	0.7	Mass spectrometric 823.8	238
Ethambutol
1	Ethambutol in human plasma	UFLC-MS	Phenomenex Gemini C18 (50 mm x 2.0 mm, 5 μm)	Acetonitrile: water (pH 2.4 adjusted with 0.5%formic acid) (80:20, v/v)	1.5	Mass spectrometric 205	239
2	Ethambutol in its dosage form and human urine	SFC-MS/MS	Inertsil ODS-C18 (100 mm x 4.6 mm, 5 μm)	Dichloromethane: methanol: formic acid (70:30:0.1, v/v/v) and supercritical CO₂	0.3 & 2	Mass spectrometric 205.1	240
Ethambutol in combination with other drugs
3	Ethambutol and Pyrazinamide in human plasma	LC-MS/MS	Chromolith SpeedROD RP-18e (50 mm x4.6 mm, 2 μm)	0.1%trifluoroacetic acid in water and 0.1% trifluoroacetic acid in methanol in gradient elution program	-	Mass spectrometric	241
Pyrazinamide
1	Pyrazinamide in human plasma	LC-MS/MS	Hypersil, Gold (50 mm x 4.6 mm, 5 μm)	Methanol: 0.1 % Formic Acid in 10 mM ammonium formate (90:10, v/v)	0.4	Mass spectrometric 124.100 → 79.160	242
Pyrazinamide in combination with other drugs
2	Pyrazinamide & Isoniazid in its dosage	SFC-MS/MS	Inertsil ODS C18 (150 mm x 4.6 mm, 5 μm)	Dichloromethane: methanol: formic acid (50:50:0.1, v/v/v). Supercritical carbon dioxide (SC-CO₂)	0.3 & 2	Mass spectrometric PYZ= 130 → 60 INH= 160 → 100	243
3	Pyrazinamide (PYZ), Isoniazid (INH) and Ethambutol (EMB) in serum	LC-MS/MS	Waters C18 analytical (100 mm x 2.0 mm, 3 μm)	Acetonitrile, water and 200 mM ammonium acetate buffer pH 5.0 in gradient elution program	0.5	Mass spectrometric PYZ= 81 → 124 INH= 121 → 138.1 ETB = 116.1 → 205.1	244
4	Pyrazinamide (PYZ), Rifampicin (RIF), Isoniazid (INH), Acetyl Isoniazid (AcINH) & Ethambutol (EMB) in Human Plasma	LC-MS/MS	Gemini C18 (150 mm x 4.6 mm; 4.6 μm)	Methanol: 5 mM ammonium acetate pH 3.5 in gradient elution program	0.6	Mass spectrometric PZA: 124 / 81 RIF: 823.46 / 791.49 INH: 138.00 / 121.00 AcINH: 180 / 121 EMB: 205.16 / 116.13	245
Rifabutin in combination with other drugs
1	Rifabutin and 25-O-deacetyl Rifabutin in human plasma	LC-MS/MS	Rp (30 mm x 2.1 mm, 3 μm)	Methanol: water: acetic acid in gradient elution	-	Mass spectrometric For Rifabutin 847.5 → 815.7 For 25-O-Deacetyl Rifabutin 805.7 → 773.7	246
2	Rifabutin (RBT) & Lopinavir (LPV) in human plasma	LC-MS/MS	HS C18 (50 mm x 4.6 mm, 5 μm)	85% acetonitrile in ammonium acetate buffer (10mM, pH 4.5)	0.7	Mass spectrometric RBT= 847.7 → 815.4 LPV= 629.6 → 447.4	247
Rifapentine
1	Rifapentine in dried blood spot sample	LC-MS/MS	BEH C8 (50 mm x 2.1 mm, 1.7 μm)	5Mm ammonium formate in water and 3% DMSO in acetonitrile in gradient elution program	-	Mass spectrometric 877.6 → 845.5	248
Cycloserine
1	Cycloserine in blood plasma	HPLC/MS	Acclaim C18 (150 mm x 2.1 mm, 3 μm)	Formic acid (0.1%) and MeCN (55:45, v/v)	0.3	Mass spectrometric	249
2	Cycloserine in healthy rat blood and lung tissues	HPLC-MS/MS	C18 (150 mm x 4.6 mm, 5 μm)	Acetonitrile containing 2mM ammonium formate and 0.1% aqueous formic acid (35:65, v/v)	0.001	Mass spectrometric	250
3	Cycloserine in 50μL of human plasma	LC-MS/MS	C18	Acetonitrile & 0.5% formic acid buffer (60:40, v/v)	0.8	Mass spectrometric	251
4	Cycloserine in human plasma	LC-MS/MS	Shim-pack XR-ODS (100 mm x2.0 mm, 2.2 μm)	Methanol & 0.01% formic acid (70:30, v/v)	-	Mass spectrometric	252
p-Aminosalicylic Acid
1	p-Aminosalicylic acid	LC-MS/MS	Phenomenex Synergi Hydro-RP (150 mm x 2.0 mm, 4μm)	Methanol: 0.2% Formic acid (40:60, v/v)	0.3	Mass spectrometric 80.2 → 136.2	253
Bedaquiline
1	Bedaquiline in human serum	LC-MS/MS	HyPURITY C18 (50 mm x 2.1 mm, 3μm)	Purified water, acetonitrile, and an aqueous buffer (containing ammonium acetate [10g/liter], acetic acid [35mg/liter], and trifluoroacetic anhydride [2ml/liter] in water) in gradient elution program	0.5	Mass spectrometric 555.1 → 58.4	254
2	Bedaquiline in human plasma	HPLC-MS/MS	Agilent ZORBAX SB-18 (100 mm x 2.1 mm, 3.5 μm)	Methanol: 5mM ammonium formate(containing 0.1% foemic acid solution) (85:15, v/v)	0.3	Mass spectrometric 555.2 → 58.3	255
3	Bedaquiline in hair	LC-MS/MS	Phenomenex Synergi Polar RP (100 mm x 2.1 mm, 2.5 μm)	Water with 1% formic acid & acetonitrile with 0.4% formic acid in gradient elution program	0.3	Mass spectrometric 557.1 → 58.1	256
Delamanid
1	Delamanid in mouse plasma	LC-MS/MS	Capcell Pak C18 MG (50 mm x 2.0 mm, 3 μm)	Purified water-formic acid (1000:2, v/v) and methanol-formic acid (1000:2, v/v) in gradient elution program	0.2	Mass spectrometric 535 → 352	257
2	Delamanid in human plasma	UHPLC-MS/MS	Acquity waters BEH C18 (50 mm x 2.1 mm, 1.7 μm)	A) Ammonium bicarbonate and ammonium hydroxide in water B) Ammonium hydroxide in methanol - Use solution A & B in gradient elution program	0.5	Mass spectrometric 535.1 → 352.2	258
Pretomanid
1	Pretomanid (PA-824), Moxifloxacin (MOX) and Pyrazinamide (PYZ) in rat plasma	LC-MS/MS	An Inertsil ODS C18 (150 mm x 4.6 mm, 5 μm)	Methanol & 0.03% triethylamine in water (85:15, v/v)	0.5	Mass spectrometricPA-824= 360.1→175.0 MOX= 402.1→260.0 PYZ= 81.2	259
1^st Line Anti-TB
1	Pyrazinamide, Rifampicin, Isoniazid, & Ethambutol	HPLC-MS/MS	C18	Methanol in 0.3% formic acid and water in gradient elution program	-	Mass spectrometric	260
2	Pyrazinamide, Rifampicin, Isoniazid, & Ethambutol	UPLC-MS/MS	Acquity UPLC HSS T3 (150 mm x 2.1 mm, 1.8 μm)	Water + 0.05% of formic acid and Acetonitrile + 0.05% of formic acid in gradient elution program	-	Mass spectrometric	261
3	Pyrazinamide, Rifampicin, Isoniazid, & Ethambutol in human plasma & PBMCs	UPLC-MS/MS	Waters HSS T3 (150 mm x 2.1 mm, 1.8 μm)	-	-	Mass spectrometric	262
2^nd Line Anti-TB
1	Nine second-line anti-tuberculosis drugs	UPLC-MS/MS	Waters HSS T3 column (50.0 mm x 2.1 mm, 1.8 μm)	10 mM ammonium formate in 0.1% formic acid and acetonitrile in 0.1% formic acid in gradient elution program	0.2	Mass spectrometric	263

B. Capillary Electrophoresis:

TABLE 13: CAPILLARY ELECTROPHORESIS

S. no.	Drug /Sample	Description	Ref. No.
Isoniazid
1	Isoniazid (INH)	Capillary electrophoresis method coupled with chemiluminescent (CL) detection was proposed for the analysis of isoniazid based on the enhancement effect of INH to CL emission of luminol-periodate potassium reaction. Under the optimal conditions, INH can be assayed in the range of 7.0 × 10^-7 to 3.0 × 10^-5 g/mL (R (2) = 0.9990) with a limit of detection of 3.0 × 10^-7 g/mL (signal-to-noise ratio of 3). The whole analysis process can be completed within 2.5 min with a theoretical plate number of 6258.	264
Ethambutol
1	Ethambutol (EMB)	CE with capacitively coupled contactless conductivity detection. The separation of EMB and its main product of degradation were achieved in less than 3 min with a resolution of 2.0. Using the best separation conditions, linearity of 0.9976 (R², five data points), the sensitivity of 1.26x10^-4 V min µmol/L, and LOD and quantification of 23.5 and 78.3 µmol/L, respectively, were obtained.	265
Rifabutin
1	Rifabutin and human serum albumin in pharmaceutical formulations	Capillary zone electrophoresis (CZE) was used for simultaneous determination of rifabutin and human serum albumin. CE conditions: a quartz capillary tube (internal diameter 75mm, effective length 50cm, total length 60cm), the capillary temperature was 25°С, the voltage applied to the capillary tube was +20kV, the UV detection wavelength was 214nm, hydrodynamic injection of the sample was performed at 30mbar for 5s, tetraborate buffer solution (0.01М, рН9.2). The obtained results are characterized by high efficiency (number of theoretical plates up to 260,000) and sufficient sensitivity (LOQ starting from 0.02μg/ml for RFB).	266
p-Aminosalicylic Acid
1	p-Aminosalicylic acid and its N-acetylated metabolite in human urine	A capillary zone electrophoresis method has been developed for the determination of p-amino salicylic acid (PAS) and its metabolite, N-acetyl-p-aminosalicylic acid (N-acetyl-PAS), in urine. A good separation of the analytes is achieved in a run time of 12 min (15 min total, including capillary wash). A linear relationship was observed between time-normalized peak area and the concentration of the parent and metabolite with correlation coefficients greater than 0.9990.	267

C. Flow Injection Analysis:

TABLE 14: FLOW INJECTION ANALYSIS

S. no.	Drug /Sample	Description	Ref. No.
Ethambutol
1	Ethambutol in synthetic urine	FIA using a graphite-polyurethane composite electrode as an amperometric detector. In order to characterise the electrochemical behaviour of ethambutol at pH = 8.0 voltammetric studies were performed. The detector was assembled in a flow injection apparatus and operated at +1.2 V (vs. Ag/AgCl (NaCl sat.)). The linear response for the method was extended up to a 1.1 mmol L^-1 ethambutol solution with a detection limit of 0.0634 mmol L^-1. The reproducibility of current responses for injections of 0.7 mmol L^-1ethambutol solution was evaluated to be 5.1% (n = 30) and the analytical frequency was 161 determinations h^-1.	268
p-Aminosalicylic Acid
1	p-Aminosalicylic acid derivatives	FIA with spectrophotometric detection (λ 510 nm). The best conditions were attained using a mixture of ethanol (methanol) and a buffer solution of pH 6.68 (30: 70 vol %). The analytical range for the analytes was 0.08-5.0 μg/ml.	269

D. Chemiluminescence Method:

TABLE 15: CHEMILUMINESCENCE METHOD

S. no.	Drug /Sample	Description	Ref. No.
Rifampicin
1	Rifampicin	Rifampicin can enhance the chemiluminescence (CL) of peroxomonosulfate‐cobalt (II) system, and the CL intensity is strongly dependent on the rifampicin concentrations. Based on this phenomenon, a rapid and sensitive flow injection CL method was developed for the determination of rifampicin. The relative CL intensity was linear with the rifampicin concentration over the range of 5×10 to 1×10 g/mL (r=0.9991), the detection limit was 7×10 g/mL (S/N=3), and the relative standard deviation was 2.7% for 6×10 g/mL rifampicin (n=11).	270

CONCLUSION: From all information given in the analytical review, it can be concluded that various UV-Visible spectrophotometric, Spectrofluori-metric, High-performance liquid chromatography (HPLC), High-performance thin layer chromato-graphy (HPTLC), Gas chromatography (GC), Micellar electro-kinetic capillary chromatography, Electrochemical, Titrimetric, Liquid chromatography / Mass spectrometry (LC/MS), Capillary electro-phoresis, Flow injection analysis, and Chemilumi-nescence were used for the determination of the first line, oral second line and newer anti-TB drugs alone and in combination. These methods have been successfully used on a routine basis and allow the quantification of the drugs in raw materials, pharmaceutical formulations, and biological matrices in a short analytical time. These all methods are sensitive, simple, fast, accurate, and reproducible, as well as possess excellent linearity & precision characteristic. These observations make it possible to anticipate the use of these methods in future analytical research work for Anti-TB drugs.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

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How to cite this article:

Jani RJ and Patel PU: Review: available analytical methods for the estimation of first line, second line oral and newer anti-TB drugs. Int J Pharm Sci & Res 2021; 12(5): 2500-34. doi: 10.13040/IJPSR.0975-8232.12(5).2500-34.

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Language: English

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Authors: R. J. Jani * and P. U. Patel

Authors Address: Department of Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Mehsana, Gujarat, India.

Email: janiriddhi52@gmail.com

Received: 25 April 2020

Revised: 10 November 2020

Accepted: 12 April 2021

DOI: 10.13040/IJPSR.0975-8232.12(5).2500-34

Published: 01 May 2021

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International Journal Of
Pharmaceutical Sciences And Research

An International Journal published monthly
An Official Publication of Society of Pharmaceutical Sciences and Research

REVIEW: AVAILABLE ANALYTICAL METHODS FOR THE ESTIMATION OF FIRST LINE, SECOND LINE ORAL AND NEWER ANTI-TB DRUGS

Article Information

International Journal Of Pharmaceutical Sciences And Research

An International Journal published monthly An Official Publication of Society of Pharmaceutical Sciences and Research

REVIEW: AVAILABLE ANALYTICAL METHODS FOR THE ESTIMATION OF FIRST LINE, SECOND LINE ORAL AND NEWER ANTI-TB DRUGS

Article Information

International Journal Of
Pharmaceutical Sciences And Research

An International Journal published monthly
An Official Publication of Society of Pharmaceutical Sciences and Research