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“RP-HPLC METHOD DEVELOPMENT & ITS VALIDATION FOR
SIMULTANEOUS ESTIMATION OF ANTI-TUBERCULOSIS DRUG IN
HUMAN PLASMA”
MASTER OF PHARMACY
(PHARMACEUTICAL CHEMISTRY)
SUPERVISED BY : SUBMITTED BY:
Dr. DEEPTI JAIN ANAND SHRIVASTAVA
SCHOOL OF PHARMACEUTICAL SCIENCES
RAJIV GANDHI PROUDYOGIKI VISHWAVIDYALAYA
BHOPAL
2016
1

CONTENTS
 Introduction
 Literature Review
 Drug Profile
 Research Envisaged
 Plan of work
 References
2

INTRODUCTION
 Tuberculosis is an infectious bacterial disease characterized by the
growth of nodules (tubercles) in the tissues, especially the lungs.
 Tuberculosis, or TB caused by Mycobacterium tuberculosis. It is
transmitted from person to person via droplets from the throat and
lungs of people with the active respiratory disease.
 The symptoms of active TB of the lung are coughing, sometimes
with sputum or blood, chest pains, weakness, weight loss, fever and
night sweats.
 Tuberculosis is treatable with a six-month course of antibiotics.
3

Global situation and trends:
Tuberculosis (TB) is contagious and airborne. It ranks alongside HIV
as a leading cause of death worldwide. 9.6 million people are
estimated to have fallen ill with TB in 2014: (5.4 million men, 3.2
million women and 1.0 million children). An estimated 1.2 million
people living with HIV developed TB in 2014. There have been major
advances in prevention, diagnosis and treatment of TB: mortality has
fallen 47% since 1990. Effective diagnosis and treatment of TB saved
an estimated 43 million lives between 2000 and 2014. TB remains one
of the world’s biggest threats. In 2014, TB killed some 1.5 million
people (1.1 million HIV-negative and 0.4 million HIV-positive).
4

 Most common TB drugs
o Isoniazid
o Rifampin
o Ethambutol
o Pyrazinamide
 For drug-resistant TB, a combination of antibiotics called
fluoroquinolones and injectable medications, such as amikacin,
kanamycin or capreomycin, are generally used.
5

BIOANALYSIS
 Bioanalytical methods are used for the quantitative analysis of drugs
and their metabolites in the biological media like saliva, urine,
plasma, serum.
 Development and validation of bioanalytical method is important to
understand the pharmacokinetics of any drug and/or its metabolites.
 Bioanalytical method development consists of three essential inter
related parts sample preparation, chromatographic separation and
detection by using proper analytical method.
 Validation of a Bioanalytical method is the process by which it is
established that the performance characteristics of the method meet
the requirements for the intended Bioanalytical application.
6

BIOANALYTICAL METHOD VALIDATION
Bioanalytical method validation is the approach employed to indicate
that the analytical method used to assess an analyte in biological matrix
is reliable and also reproducible.
 Accuracy
 Selectivity
 Precision
 Specificity
 Detection limit
 Quantitation limit
 Linearity
 Range
 Robustness.
7

LITERATURE REVIEW
S. NO. METHOD DESCRIPTION
1 A simple and rapid
HPLC/UV method for
simultaneous quantification
for four constituents in Anti-
Tuberculosis 4-fdc tablets by
pre-column derivatization
Mobile Phase : Mobile phase gradient consisting of Acetonitrile- Phosphate Buffer
(8 mM, pH – 6.8)
Column : Phenomenex Luna C18 (250 X 4.6 mm I.D.)
Mode of Method: Gradient Mode
UV Wavelength : 210 nm
Injection Volume : 20 µL
Flow Rate : 1 ml/min
Retention Time (in min.) : PYP (4.65), INH (13.14), RIF (18.01), EMB (21.66)
2 Development and
validation of HPLC
method for the
determination of
pyrizanamide in human
plasma
Mobile Phase : Mixture of 15:85 % v/v Methanol and 0mM potassium
dihydrogen phosphate (pH adjust to 7.4)
Column: Phenomenox ODC2 C18 Column (150 X 4.6 mm I.D.) 5µm
Mode of Method : Isocratic Mode
Retention Time (in min.) :PZA (6.80)
8

3 Development and
validation of RP-HPLC
method for simultaneous
estimation of Rifampicin,
Isoniazid, pyrizinamide in
human plasma
Mobile Phase: Mixture of ACN, Methanol and Water in the ratio of 30:5:65 v/v
pH adjust to 5.2
Column : Phenomenox ODC2 C18 Column (150 X 4.6 mm I.D.) 5µm
Retention Time (in min.) : RIF (5.99), INH (5.11), PZA (10.97)
4 Development and
validation of HPLC
method for determination
of Rifampicin in human
plasma
Mobile Phase : Mixture of 42:60 & v/v Acetonitrile and 10mM potassium
dihydrogen phosphate (pH adjust to 3.2)
Column : Phenomenox ODS2 C18 Column (150 X 4.6 mm I.D.) 5µm
9

5 Development and
method for quantitative
estimation of Pyrazinamide
in bulk and
pharmaceutical dosage
forms.
Mobile Phase : Phosphate buffer (pH 4.4): Methanol 80:20 (v/v)
Column : Hypersil C8 (4.6 x 250mm, 3.5 μm) column
Retention Time (in min.) :PYP (3.62)
6 Method deveopment and
validation of anti-
tubercular drugs in fixed
dose formulation by RP-
HPLC technique”
Mobile Phase : Methanol : ammonium acetate buffer (pH-7.03) in the ratio of
(50:50).
Column : C18 Thermo Hypersil ODS, (250 X5.4 mm X 4.5μ) column
Flow Rate : 1.3 ml/min
Retention Time (in min.) : ETH and INH were 2.01 min and7.0 min
respectively.
10

7 Development and
method for the
determination of
pyridoxine hydrochloride,
Isoniazid, Pyrazinamide
and Rifampicin in
pharmaceutical
formulation
Mobile Phase : Acetonitrile and potassium dihydrogen phosphate buffer 11:89
v/v (pH adjusted to 4.0 ± 0.1)
Mode of Method : Gradient Mode
Retention Time (in min.) : pyridoxine hydrochloride, Isoniazid, Pyrazinamide
and Rifampicin were 3.687, 4.113, 5.041 and 12.829 min, respectively.
8 Method development and
validation for simultaneous
estimation of Isoniazid and
Ethambutol by using RP-
HPLC in bulk and
pharmaceutical dosage
form
Mobile Phase :Acetonitrile-Phosphate Buffer 65:35 v/v (pH – 4.6)
Column: Inertsil C18 (4.6 x250mm, 5μm
Injection Volume : 10µL
Retention Time (in min.) : Isoniazid and Ethambutol was found to be 2.325
and 4.322nm respectively.
11

9 Simultaneous HPLC
determination of Isoniazid
and acetylIsoniazid in
plasma
Mobile Phase : 1-hexanesulfonic acid sodium salt solution (20 mM, pH 3,
adjusted with phosphoric acid) and acetonitrile
Column : Waters X-terra RP18 column
Retention Time (in min.) : 4.5 and 7.4 min for INH and AcINH
10 Contribution to the
development and
validation of a high
performance liquid
chromatography by the
UV detection method for
Isoniazid and omeprazole
determination
Mobile Phase : Triethylamine (with a pH value of 10.5): acetonitrile (67:33,
v/v)
Column: C8 column Octasilil (Purospher RP8) 250mm x 4.6 mm i.d. 5μm
Retention Time (in min.): 2.323 min for Isoniazid; 3.497 min for 2-
pyridylamine; 4.013 min for omeprazole and 6.837 min, respectively.
12

11 Validation of HPLC
methods for determination
of Isoniazid, Rifampicin,
Pyrazinamide, and
Ethambutol in a fixed-dose
combination
antituberculosis
Mobile Phase : Mobile phase gradient consisting of Acetonitrile- Phosphate
Buffer (8 mM, pH – 6.8)
Injection Volumev : 20 µL
Retention Time (in min.) : PYP (4.65), INH (13.14), RIF (18.01), EMB (21.66)
12 A validated high
performance liquid
chromatography method
for the determination of
Rifampicin and desacetyl
Rifampicin in plasma and
urine
Mobile Phase : Mobile phase was 0.05 M potassium dihydrogen phosphate
buffer (pH 2.6): acetonitrile (55:45 v/v)
Column: Phenomenex Luna C18 (250 X 4.6 mm I.D.)
Retention Time (in min.) : DRIF, RIF and Rifapentine (RPN), the internal
standard were 2.9, 4.8 and 10.5 min respectively.
13

DRUG PROFILE
ETHAMBUTOL
 Molecular Structure :
 Molecular Formula : C10H24N2O2
 Molecular Weight : 204.31 g·mol−1
 Solubility : Soluble in water, DMSO; sparingly soluble in
ethanol; difficult to dissolve in acetone and
chloroform
 Pka : 6.6 & 9.5
 Dosage : Available as 100 and 400 mg tablets. 15 mg/kg
daily or up to 25 mg/kg but risk of ocular toxicity.
Weekly dose, 30 mg/kg three times/week.
 Plasma Half Life(t½) : 3–4 hours
 Protein Binding : 20 – 30%
 Mechanism of Action : Ethambutol inhibits arabinosyl transferases which
is involved in cell wall biosynthesis. By inhibiting this enzyme, the bacterial cell
wall complex production inhibited. This leads to an increase in cell wall
permeability.
14

ISONIAZID
 Molecular Formula : C6H7N3O
 Molecular Weight : 137.13928 g/mol
 Solubility : Solubility in water, ethanol, Chloroform, Practically insoluble
in ether, benzene
 Pka : 1.82(at 200C)
 Dosage : Adult dosing generally 300 mg capsule administered orally,
once daily; or 15 mg/kg up to 900 mg/day, two or three
times/week, ideally dose administered one hour before or two
hours after a meal.
 Plasma Half Life(t½) : Fast acetylators: 0.5 to 1.6 hr. Slow acetylators: 2 to 5 hr.
 Protein Binding : 0 – 10%
 Mechanism of Action : Isoniazid is a prodrug and must be activated by bacterial
catalase. Specficially, activation is associated with reduction of the mycobacterial ferric
KatG catalase peroxidase to form an oxyferrous enzyme complex. Once activated, isoniazid
inhibits the synthesis of mycoloic acids, an essential component of the bacterial cell wall.
actively growing intracellular and extracellular Mycobacterium tuberculosis organisms.
Specifically isoniazid inhibits InhA, the enoyl reductase from the NAD cofactor. It is the
INHNAD. adduct that acts as a slow, tight binding competitive inhibitor of InhA.
15

PYRAZINAMIDE
 Molecular Formula : C5H5N3O
 Molecular Weight : 123.11 g/mol
 Solubility : Soluble in chloroform, methylene chloride; less soluble in
benzene; sparingly soluble in water
 Pka : -0.5
 Dosage : Dose 20- 25 mg/kg daily, or 50-70 mg/kg three times in a
week.
 Plasma Half Life(t½) : 9-10 hrs
 Protein Binding : ~10%
 Mechanism of Action : Pyrazinamide diffuses into M. tuberculosis, where the
enzyme pyrazinamidase converts pyrazinamide to the active form pyrazinoic acid. Under
acidic conditions, the pyrazinoic acid that slowly leaks out converts to the protonated
conjugate acid, which is thought to diffuse easily back into the bacilli and accumulate.
The net effect is that more pyrazinoic acid accumulates inside the bacillus at acid pH
than at neutral pH. Pyrazinoic acid was thought to inhibit the enzyme fatty acid synthase
(FAS) I, which is required by the bacterium to synthesise fatty acids.
16

RIFAMPICIN
• Molecular Structure :
• Molecular Formula : C43H58N4O12
• Molecular Weight : 822.94 g/mol
• Solubility : Freely soluble in chloroform and DMSO; soluble in
ethyl acetate, methanol, tetrahydrofuran; slightly
soluble in acetone, water, carbon tetrachloride
• Pka : 1.7
• Dosage : Dose 10 mg/kg, in a single daily administration, not
to exceed 600 mg/day, oral or i.v.
• Plasma Half Life(t½) : 3.35 (±0.66) hours
• Protein Binding : 89%
• Mechanism of Action : Rifampin inhibits the DNA dependent RNA
polymerase, leading to a suppression of RNA synthesis and cell death.
17

RESEARCH ENVISAGED
 Bioanalytical method employed for the quantitative
determination of drugs and their metabolites in
biological fluids plays a significant role in the evaluation
and interpretation of bioavailability, bioequivalence,
pharmacokinetic, and toxicokinetic study data.
 Monitoring the concentration of the drug in the blood
and plasma ascertains that the calculated dose actually
delivers the plasma level required for the therapeutic
effect.
18

 To develop selective and sensitive analytical method for
the quantitative evaluation of drug and their metabolism
are critical for the successful conduct of
biopharmaceutics and clinical pharmacology studies.
 The manuscript could be used as a guide in some
Therapeutic Drug Monitoring, Bioavailability and
Bioequivalence studies of drug candidates.
 Literature survey reveals that as such no UV and HPLC
method has yet been reported for simultaneous
estimation of Isoniazid, Rifampin, Ethambutol and
Pyrazinamide.
19

PLAN OF WORK
 Literature Survey
 Development of Method
• Selection of suitable detecting wavelength.
• Selection of optimization of method for extracting drug for plasma.
• Determination of appropriate working concentration range.
• Validation of proposed method as per ICH guidelines.
• Analysis of biological samples with the developed method,
• Recovery study of statistical evaluation of developed method.
 Validation of Developed Method
20

REFERENCES
 Tuberculosis Fact Sheet No. 104, World Health Organization November
2010. Retrieved 26 July 2011.
 Robertson Brian D, Brennan Patrick J, Young Douglas B, Handbook of
Anti-Tuberculosis Agents, 2008 88(2): 85–170
 Wong EB, et al. Rising to the challenge: new therapies for tuberculosis.
Trends in Microbiology. 2013,21:493
 Tripathi K.D, Essentials of Medical Pharmacology, 4th Edition, Jaypee
Brothers Medical Publishers (P) Ltd, New Delhi Page No. 475
 US Food and Drug Administration, Guidance for industry- Bioanalytical
method validation, Center for Drug Evaluation and Research, Rockville,
MD, 2001.
 "Ethambutol” (CHEBI:4877) Chemical Entities of Biological Interest. UK:
European Bioinformatics Institute. 18 August 2010. Main. Retrieved 26
April 2012. (Accessed December 24,2015.)
 http://www.drugbank.ca/drug/DB00951 (Accessed December 24,2015.)
 http://www.drugbank.ca/drug/DB00339 (Accessed December 24,2015.)
21

 ICH, Specifications test procedures and acceptance criteria for new drug
substances and new drug products: chemical substances. International
Conference on Hormonisation, IFPMA, Geneva 1999
 Chung Chow Chan, Analytical Method Validation: Principle and Practices.
(727-742).
 U.S. Food and Drug Administration. Guidance for Industry,Bioanalytical
Method Validation, September 2013:1
22

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