Download Lui J, Sun J and his co-workers carried out the HPLC determination

“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ANTI
TUBERCULOSIS DRUGS BY HPLC”
a) BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Assay is a test to determine the composition, the content of the ingredients, and
the quantitation limit unit of medicine by physical, chemical, or biological procedures. The
study focuses on validation of the quantitative assay by high performance liquid
chromatography (HPLC).
Analytical method development and validation involves a series of activities that
are ongoing during the life cycle of a drug product and drug substance.1
Method development
Method validation/revalidation
QC laboratory
Analytical method development should be performed to the extent that it is
sufficient for its intended purpose.
Upon successful completion of method development, the analytical method will
then be validated to show proof that it is suitable for its intended purpose.
Tuberculosis treatment refers to the medical treatment of the infectious disease
tuberculosis. Active tuberculosis will kill about two of every three people affected if left
untreated.
The scope and need of the present project is to develop and validate an analytical
method for a drug to detect its potency as it is the major criteria for any drug to posses.
HPLC method is being used because it is the preferred method for development
and validation in the recent trend, and validation of a drug is regulatory requirement and also
it ensures that the products and process are fit for their intended use.
6.2 Review of the literature:
Ali J, Ali N and his co-workers developed and evaluated a stability-indicating HPTLC
method for analysis of anti tubercular drugs. In this method the drug combination, isoniazid
and rifampicin was analysed by developing HPTLC technique. Analysis was done in both
bulk drugs and formulations. In this method compounds were separated by using aluminium
backed silica gel 60 F254 plates. Densitometric analysis of isoniazid and rifampicin was
performed at 254 nm. The developed method was validated and accuracy, specificity,
linearity, range, quantitation limit were determined. They reported this method is easy,
sensitive, simple method2.
Kumar P and Sreeramulu J developed and validated stability indicating ReversedPhase High Performance Liquid Chromatography Method for assay of Prothionamide in Pure
and Pharmaceutical Dosage form. They developed a new technique which was a stability
indicated RP-HPLC method for the analysis of Prothionamide. It is an anti tubercular drug.
The column used was a RP C18 column and mobile phase used was Methanol: Buffer
solution and PH was adjusted to 4.5. As drug undergoes degradation in different conditions
like acidic, basic, photochemical degradation and thermal degradation all the peaks of degradation product were differentiated by retention time. This method was validated for
robustness, recovery, ruggedness and precision3.
Evaluation of the recently reported USP gradient HPLC method for analysis of antituberculosis drugs for its ability to resolve degradation products of rifampicin was carried out
by Mohan B, Sharda N and Singh S. In this method they determined the ability of a recently
notified USP HPLC method quantitative determination of rifampicin, isoniazid and
pyrazinamide in fixed dose combination (FDC) formulations to resolve major degradation
products of rifampicin. Resolving power depends on the make of the column. This study gave
a good insight into the performance of the proposed USP method for the assay of Rifampicin,
Isoniazid and pyrazinamide in pharmaceutical FDC formulations4.
Karthikeyan K, Arularasu GT and his co-workers developed and validated an indirect
RP-HPLC method for enantiomeric determination of d-cycloserine drug substance. In this
article a new RP-HPLC method was developed by using chiral derivatization reagents like o-
phthalaldehyde and N-acetyl-l-cysteine. In this Zorbax SB Phenyl HPLC column was used
for resolving diastereomers. A mobile phase of 95:05 (v/v), 20mMNa2HPO4 (pH 7), and
acetonitrile was used and the flow rate was maintained at 1.0ml/min. The above developed
method was validated and suitability was determined5.
Comparison of the data regarding two HPLC methods for the determination of
isoniazid. In this method they developed two HPLC methods for determination of Isoniazid.
Waters 2695 liquid chromatography and a UV - Waters 2489 detector were used for this
experiment. The first method (I) used a Nucleosil 100-10 C18 column (250 x 4.6 mm), a
mobile phase formed by a mixture of acetonitrile/10-2 M oxalic acid (80/20) and a flow rate
of 1.5 ml/ min; detection was done at 230 nm. The second method (II) used a Luna 100-5
C18 column (250 x 4.6 mm), a mobile phase formed by a mixture of methanol/acetate buffer,
pH = 5.0 (20/ 80), and a flow rate of 1 ml/min; detection was done at 270 nm. Both methods
were validated and applied for INH determination6.
Lui J, Sun J and his co-workers carried out the HPLC determination of rifampicin and
related compounds in pharmaceuticals using monolithic column. They developed a HPLC
method by using c18 monolithic column. In this method they analysed rifampicin and four
related compounds. They used methanol-acetonitrile-monopotassium phosphate (0.075 M)citric acid (1.0M) as the mobile phase at a flow rate of 2 ml/min. The total run time was less
than 11 min. The developed method was validated and accuracy and precision were
determined7.
Review of literature reveals the determination of contents and dissolution of
ethambutol hydrochloride in fixed dose combinations for anti tuberculosis drugs by high
performance liquid chromatography with pre-column derivatization. In this RP-HPLC
method derivatisation should be carried out in non aqueous environment. ethambutol was
dervatised with phenylethylisocynate in a molar ratio of 1:6. Active recovery of the sample
was 98.7% and standard deviation was 0.70%. the linear regression of standard calibration
curve was good. The method was good rapid, specific and sensitive, so it was successfully
applied for dissolution and quantitisation of ethambutol in fixed dose combinations (FDCs)8.
Validation of a RP-LC method for the simultaneous determination of isoniazid,
pyrazinamide and rifampicin in a pharmaceutical formulation was carried out by Calleri E,
De Lorenzi E. A simple accurate liquid
chromatographic method was developed and
validated for the estimation of isoniazid, pyrazinamide and rifampicin in combined dosage
forms. The drugs were chromatographed on a reverse phase c18 column using a mobile phase
gradient and monitored at corresponding maximum peaks with rention time compared to
standards and confirmed with charcterstic spectra of diode array detector solutions.
Concentrations were measured on weight basis to avoid using of internal standard. The
method does not require any sample preparation except preparation of guard column. The
method is simple rapid specific. Due to its simplicity, and accuracy the method is used in
routine quality control of anti tuberculosis combined dosage forms9.
Butterfield AG, Lovering EG and Sears RW, have carried out the simultaneous
determination of isoniazid and 1-isonicotinyl-2-lactosylhydrazine (I) in isoniazid tablet
formulations by using high performance liquid chromatographic. An aliquot of a diluted
aqueous tablet extract was introduced onto a micro particulate cyanopropyl bonded-phase a
valve-loop injector was used as a column and acetonitrile-0.01 M, pH 3.5 aqueous acetate
buffers (5:95) used as a mobile phase. By this method the compound can be determined at
levels as low as 0.5% of the isoniazid label claim10.
Gaitonde CD and Pathak PV developed a rapid and sensitive method for the
estimation of isoniazid, pyrazinamide and rifampicin in combined dosage form by reversedphase liquid chromatography. In this method the sample was dissolved in the mobile phase
and the mixture was filtered. A portion of the column analysed by HPLC by using column
(25 cm × 4.6 mm) of Excalibur ODS-CN (5 µm). In this method they used 5mN-tetrabutyl
ammonium hydroxide (adjusted to pH 3 with phosphoric acid) - methanol (1:4) as the mobile
phase and detection was carried out at 265 nm. The calibration graph was rectilinear for 0.1
to 0.5 mg/ml of isoniazid, pyrazinamide and rifampicin11.
6.4 OBJECTIVE OF STUDY:
The method development of the HPLC technique involves the following steps:
 Step 1: Define method objectives and understand the chemistry.
Determine the goals for method development and to the understand the chemistry of
the analytes and drug products.
 Step 2: Initial HPLC conditions
Develop preliminary HPLC conditions to achieve minimally acceptable separations.
These HPLC conditions will be used for all subsequent method development
experiments.
 Step 3: Sample preparation procedure
Develop a suitable sample preparation scheme for the drug product.
 Step 4: Standardization
Determine the appropriate standardization method and the use of relative response
factors in calculations.
 Step 5: Final method optimization/robustness
Identify the “weaknesses” of the method and optimize the method through
experimental design. Understand the method performance with different conditions,
different instrument set ups and different samples.
 Step 6: Method validation
Complete method validation according to ICH guidelines.
Assay procedures are intended to measure the analyte present in a given sample.
In the context of this document, the assay represents a quantitative measurement of the major
component in the drug substance. For the drug product, similar validation characteristics also
apply when assaying for the active or other selected component(s). The same validation
characteristics may also apply to assays associated with other analytical procedures (e.g.,
dissolution).
The objective of the analytical procedure should be clearly understood since this
will govern the validation characteristics which need to be evaluated. Typical validation
characteristics which should be considered are listed below:
6.4.1. Analytical Procedure
The analytical procedure refers to the way of performing the analysis. It should describe in
detail the steps necessary to perform each analytical test. This may include but is not limited
to the sample, the reference standard and the reagents preparations, use of the apparatus,
generation of the calibration curve, use of the formulae for the calculation, etc.
6.4.2. Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components,
which may be expected to be present. Typically these might include impurities, degradants,
matrix, etc.
Lack of specificity of an individual analytical procedure may be compensated by
other supporting analytical procedure(s).
This definition has the following implications:
Identification: to ensure the identity of an analyte.
Purity Tests: to ensure that all the analytical procedures performed allow an accurate
statement of the content of impurities of an analyte, i.e. related substances test, heavy metals,
residual solvents content, etc.
Assay (content): to provide an exact result this allows an accurate statement on the content of
the analyte in a sample.
6.4.3. Accuracy
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value and
the value found. This is sometimes termed trueness.
6.4.4. Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions. Precision may be considered at three
Levels: repeatability, intermediate precision and reproducibility. Precision should be
investigated using homogeneous, authentic samples or by using artificially prepared Samples
or a sample solution.
The precision of an analytical procedure is usually expressed as the variance,
standard deviation or coefficient of variation of a series of measurements.
6.4.4.1. Repeatability
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision.
6.4.4.2. Intermediate precision
Intermediate precision expresses within-laboratories variations: different days, different
analysts, different equipment, etc.
6.4.4.3. Reproducibility
Reproducibility expresses the precision between laboratories (collaborative studies, usually
applied to standardization of methodology).
6.4.5. Detection Limit
The detection limit of an individual analytical procedure is the lowest amount of analyte in a
Sample which can be detected but not necessarily quantitated as an exact value.
6.4.6. Quantitation Limit
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in
a sample which can be quantitatively determined with suitable precision and accuracy. The
quantitation limit is a parameter of quantitative assays for low levels of compounds in sample
matrices, and is used particularly for the determination of impurities and/or degradation
product.
6.4.7. Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the sample.
6.4.8. Range
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which it
has been demonstrated that the analytical procedure has a suitable level of precision, accuracy
and linearity.
6.4.9. Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides an indication of its
reliability during normal usage.
b) Materials and methods:
7.1 Source of data:
Data will be obtained from drug bank, Rx list, Pub med, Science Direct, Medline, US
patent office website and other Internet facilities, literature search, and related articles from
library of Krupanidhi College of Pharmacy.
7.2 Method of collection of data (including sampling procedures if any):
Data will be collected from the following step wise experimental procedures proposed in
the study:
This is to verify that the method performance is not affected by typical changes in
normal experiments. Therefore, the variation in method conditions for robustness should be
small and reflect typical day-today variation.
1. HPLC conditions
a. HPLC column (lot, age, brand)
b. Mobile-phase composition (pH ± 0.05 unit)
c. HPLC instrument (dwell volume, detection wavelength ± 2 nm, column temperature ±
5◦C, flow rate)
2. Sample preparation
a. Sample solvent (pH ± 0.05 unit)
b. Sample preparation procedure (shaking time, different membrane filters)
c. HPLC solution stability
Built-in Robustness in Method procedure:

Weighing error

Dilution error

Mobile phase as sample solvent

Buffer

Isocratic method

Sonication
7.3 Does the study require any investigation or interventions to be conducted on
patients or other humans or animals?
No.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Not Applicable
c) List of References:
1.
ICH Harmonized Tripartite Guideline, ICH Q2A, Text on Validation of Analytical
Procedures [Internet] 1993 Oct 26 [updated 1994 Oct 27]. Available from: URL:
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
2.
J Ali, N Ali, Y Sultana, S Baboota, and S Faiyaz. Development and validation of a
stability-indicating HPTLC method. Acta Chromatogr 2007:168-70.
3.
Praveen kumar M and Sreeramulu J. Development and validation of a Stability
indicating Reverse Phase High Performance Liquid Chromatography Method for assa
y of Prothionamide in Pure and Pharmaceutical Dosage form. J Pharm Biomed Anal
2002 Aug; 29(6):1089-96.
4.
Mohan B, Sharda N and Singh S. Evaluation of the recently reported USP gradient
HPLC method for analysis of anti-tuberculosis drugs for its ability to resolve
degradation products of rifampicin 2003 Mar; 31(3):607-12.
5.
Karthikeyan K, Arularasu GT, Ramadhas R and Pillai K.
Development and
validation of indirect RP-HPLC method for enantiomeric purity determination of dcycloserine drug substance. J Pharm Biomed Anal 2011 Mar 25; 54(4):850-4.
6.
Garbulet D, Spac AF and Dorneanu V. Comparative data regarding two HPLC
methods for determination of isoniazid. Rev Med Chir Soc Med Nat Iasi 2009 OctDec; 113(4):1285-8.
7.
Liu J, Sun J, Zhang W, Gao K and He Z. HPLC determination of rifampicin and
related compounds in pharmaceuticals using monolithic column. J Pharm Biomed
Anal 2008 Jan; 46(2):405-9.
8.
Sun J, Song H, Zhang T, Wang Z and He Z.
Determination of contents and
dissolution of ethambutol hydrochloride in fixed dose combinations for antitubercular drugs by high performance liquid chromatography with pre-column
derivatization. Se Pu 2006 Mar; 24(2):164-7.
9.
De LE, Furlanetto S, Massolini G and Caccialanza G. Validation of a RP-LC method
for the simultaneous determination of isoniazid, pyrazinamide and rifampicin
inCalleri E
a pharmaceutical formulation. J Pharm Biomed Anal 2002 Aug;
29(6):1089-96.
10.
Butterfield
AG,
Lovering
EG
and
Sears
RW.
High-performance
liquid
chromatographic determination of isoniazid and 1-isonicotinyl-2-lactosylhydrazine in
isoniazid tablet formulations. J Pharm Sci 1980 Feb; 69(2):222-4.
11.
Gaitonde CD and Pathak PV. Rapid and sensitive estimation of isoniazid,
pyrazinamide and rifampicin in combined dosage form by reversed-phase liquid
chromatography. Drug Dev Ind Pharm 1991; 17(9):1201-14.