Chapter 5

Materials and methods

5. Materials and Methods

INSTRUMENTS
Components of HPLC system
Chromatographic System

: Shimadzu LC-20AT

Detector

: SPD-20A Prominence UV-Vis

detector

Pump

: LC-20 AT-Shimadzu

Injector

: Rheodyne

Column

: BDS Hypersil C18, 1504.6,

3,

Software

: Spin chrome

List of Materials and Reagents

Acetonitrile (HPLC Grade)

: Merck Specialities Pvt Ltd,

Mumbai

Methanol (HPLC Grade)

: Merck Specialities Pvt Ltd,

Mumbai

Water

: Milli Q

Tetra butyl ammonium hydrogen sulphate

: Leonil Chemicals Pvt Ltd,

Bangalore

Artemether and Lumefantrine reference standards

: Ipca Pvt ltd. Mumbai

HPLC METHOD DEVELOPMENT

The parameters for the development are as follows:

Selection of detection Wavelength

Selection and optimization of chromatographic condition
Study of system suitability parameters
Application of the method to the drug in pure and pharmaceutical dosage
form.

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Chapter 5

Materials and methods

VALIDATION OF PROPOSED METHOD

The parameters for method validation are as follows:
a)
b)
c)
d)
e)
f)
g)
h)

Precision
Accuracy
Linearity or Range
Robustness
Ruggedness
Limit of detection
Limit of quantification
Assay

Development and Optimization of HPLC method

Selection of detection wavelength
The sensitivity of HPLC method that uses UV detection depends upon proper
selection of detection wavelength. An ideal wavelength is the one that gives good
response for the drugs that are to be detected. In the present study drug solution of
10g/ml was, therefore, prepared insolvent (acetonitrile). This drug solution was than
scanned in the UV region of 200-400 nm, max was determined by UV visible
spectrophotometer and the spectrum was recorded.
Optimization of chromatographic condition
Proper selection of the HPLC method depends upon the nature of the sample
(ionic or ionisable or neutral molecule), its molecular weight and solubility. The drug
selected for the present study is non-polar in nature and hence either reversed phase or
ion-pair or non-Aqueous chromatography can be used. Reversed phase HPLC was
selected for the initial separations because of its simplicity and suitability. The
standard solution of Artemether and Lumefantrine was prepared and run through the
system and different combinations of mobile phase and column were tried for
isocratic mode to get well resolved, symmetric peaks. Mobile phase was filtered
through 0.45 membrane filter. To optimize the chromatographic conditions, the
effect of chromatographic variables such as mobile phase pH, flow rate and solvent
ratio were studied. The resulting chromatograms we rerecorded and the
chromatographic parameters such as capacity factor, asymmetric factor and column
efficiency were calculated. The conditions that gave the best resolution, symmetry and
capacity factor were selected for estimation.
Method Validation

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Chapter 5

Materials and methods

The proposed method was validated as per ICH guidelines. The method was
validated in terms of parameters like linearity, accuracy, precision, ruggedness,
robustness, sensitivity etc.
Preparation of Solutions
Standard stock solution
Accurately weighed and transferred 4mg of (AT) reference standard and 24mg of
(LU) reference standard to 25ml volumetric flask, added appropriate quantity of
diluents and sonicated to dissolve it completely and the volume was made up to the
mark with the same diluents, to obtain a solution of 160g/ml of (AT) and 960g/ml
of (LU), resultant solution was ultra sonicated for5min and filtered through 0.45
membrane filter.
Preparation of working standard solution
1 ml of stock solution was pipetted out and transferred to a 10 ml volumetric
flask and made volume up to mark with diluent to get final concentration of 16g/ml
and 96g/ml for Artemether and Lumefantrine, respectively.
Buffer Preparation
Dissolve 0.33954 gm of tetra butyl ammonium hydrogen sulphate in small amount
of distilled water, transferred into 100ml volumetric flask and made up to the final
volume with HPLC gradewater. The solution was then filtered through 0.45
membrane filter and degassed.
Preparation of mobile phase
200ml (20%) of the above buffer was mixed with 800 ml of acetonitrile (80%) and
degassedinultrasonic water bath for 5 minutes and filtered through 0.45 membrane
filter.
Diluents Preparation
Mobile phase [0.01M tetra butyl ammonium hydrogen sulphate & acetonitrile
(20:80% v/v)] used as a diluent.
Validation of the proposed method
The proposed method was validated as per ICH guidelines. The method was
validated in terms of parameters like linearity, precision, accuracy, robustness,
ruggedness etc. To validate the RP- HPLC method, a series of tests were made using
the most promising conditions.
Preparation of working standard solution
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Chapter 5

Materials and methods

1 ml of stock solution was pipetted out and transferred to a 10 ml volumetric flask

and made
Volume up to mark with diluent to get final concentration of 16g/ml and 96g/ml for
Artemether and Lumefantrine, respectively.
Buffer Preparation
Dissolve 0.33954 gm of tetra butyl ammonium hydrogen sulphate in small amount
of distilled water, transferred into 100ml volumetric flask and made up to the final
volume with HPLC grade water. The solution was then filtered through 0.45
membrane filter and degassed.
Preparation of mobile phase
200ml (20%) of the above buffer was mixed with 800 ml of acetonitrile (80%) and
degassed in ultrasonic water bath for 5 minutes and filtered through 0.45 membrane
filter.
Diluent Preparation
Mobile phase [0.01M tetra butyl ammonium hydrogen sulphate & acetonitrile
(20:80% v/v)] used as a diluent.
Validation of the proposed method
The proposed method was validated as per ICH guidelines. The method was
validated in terms of parameters like linearity, precision, accuracy, robustness,
ruggedness etc. To validate the RP- HPLC method, a series of tests were made using
the most promising conditions. The solutions of the drug were prepared as per the
earlier adopted procedure given in the experiment. Solutions of the drug were
prepared as per the earlier adopted procedure given in the experiment.
a) System Suitability Test
System suitability tests are an integral part of chromatographic method. They were
used to verify the adequate reproducibility of chromatographic system for analysis. To
ascertain its effectiveness, system suitability tests were carried out on freshly prepared
standard stock solution of Artemether and Lumefantrine and the parameters like
column efficiency, resolution, peak area and tailing factor of the peaks were
calculated.
b) Linearity
Aliquot portions of standard stock solution 0.2, 0.4, 0.6, 0.8 and 1.0 ml were taken
in separate10 ml volumetric flasks. The volume was adjusted to the mark with diluent
to obtain concentrations of 3.2, 6.4, 9.6, 12.8, 16.0g/ml and 19.2, 38.4, 57.6, 76.8,
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Materials and methods

96.0, 115.2 g/ml FO Artemether and Lumefantrine, respectively. Calibration curve

was plotted over a concentration range of 3.2-16g/ml for Artemether and 19.2115.2g/ml for Lumefantrine.Calibration curve was constructed by plotting peak area
v/s concentration, the graph must be linear and the regression equation was calculated.
c) Accuracy
The accuracy of the method was determined by calculating recovery of the analyte
of interest by the standard addition method. Known amounts of working standard of
Artemether (1.6 g) and Lumefantrine (9.6 g) were added to solutions of various
concentrations like: 12.8 g, 16 g and19.2 g of Artemether and 76.8 g, 96 g and
115.2 g of Lumefantrine. Each sample was prepared in triplicate and injected. The
chromatograms were recorded and from the peak area of the drug, % recovery was
calculated.
d) Precision
The precision of the method was demonstrated by inter-day and intra-day
variation studies. The atre-day precision was evaluated by repeatedly injecting (n=5)
standard solutions of Artemether (16g/ml) and Lumefantrine (96g/ml). Similarly,
the inter-day precision was evaluated in five consecutive days. The Artemether and
Lumefantrine concentrations were determined and the relative standard deviations
(R.S.D.) were calculated.
e) Sensitivity
Limit of detection (LOD) and quantification (LOQ) were estimated from the
signal-to-noise ratio. The LOD and LOQ were calculated by the use of the equations:
LOD = 3.3 / S
LOQ = 10 / S
Where is the standard deviation of intercept of calibration plot and S is the
average of the slope of the corresponding calibration plot.
f) Ruggedness
The Ruggedness of an analytical method is the degree of reproducibility of test
results obtained by the analysis of the same samples under a variety of normal test
conditions, such as different laboratories, different analysts, different instruments,
different lots of reagents, different elapsed assay times, different days, etc.
Ruggedness is normally expressed as the lack of influence on test results of operations
and environmental variables of the analytical method. Sample solutions of Artemether
(16g/ml) and Lumefantrine (96 g/ml) were prepared and analyzed by using same
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operational and environmental conditions. Ruggedness of the proposed method was

determined by analyzing sample by different analyst.
g) Robustness
To evaluate robustness of the developed method, few parameters like mobile
phase ratio, wavelength, flow rate, etc. were deliberately varied. It was observed that
there were no marked changes in chromatograms, which demonstrated that the
developed RP-HPLC method is robust. Sample solutions of Artemether (16g/ml) and
Lumefantrine (96 g/ml) were prepared and analyzed by changing the flow rate and
wavelength.
Assay
Preparation of sample solution
Five Tablets of Artemether and Lumefantrine were weighed and finely powdered.
A quantity equivalent to 4mg of Artemether and 24mg of Lumefantrine was
transferred into 25 mlvolumetric flask and appropriate amount of diluent was added.
The contents were sonicatedtodissolve completely and the volume was made up to the
mark with diluent and filtered through0.45m membrane filter. 1 ml of stock solution
was pipetted out and transferred to a 10 ml volumetric flask and made volume up to
mark with diluents to get final concentration of 16g/ml and 96g/ml for Artemether
and Lumefantrine, respectively.
Preparation of working standard solution
1 ml of stock solution was pipetted out and transferred to a 10 ml volumetric
flask and made

volume up to mark with diluent to get final concentration of

16g/ml and 96g/ml for Artemether and Lumefantrine, respectively. Twenty micro
liters of sample and standard solutions were injected in HPLC in triplicate and
chromatograms were recorded.

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