Page 1 Chapter 1

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Chapter 1. Introduction
HPLC-METHOD DEVELOPMENT
HPLC method is used to analyze several o drugs because of several advantages
of this method used by specific, accurately, preciseing, rapid, automatically
and eliminates tedious extraction and isolation procedures.
Advantages:
1. Speed (analysis sample willmbe be accomplished in 20 mins or less)
2. Greater aquired sensitivity
Role of the Column
The HPLC colums are the total heart of the methods, its difficult in performing
the sample separation. The columns should possess the selectively, efficiency,
and reproducibility .All of these characteristic are dependent on the column
manufactures release of better quality columns and packaging materials. .
Commonly used reversed phases are C18 (octadecylsilane, USP L1),
C8(octylsilane, USP L7), phenyl (USP L11), and cyano (USP L18) (24).
Initial Mobile Phase Selection:
Mobile phase is the most important parameter in reversed-phase HPLC. Mobile
phase selection is the 2nd final common step in the develop of the clearence
method and first developed one is the selection and of the adsorbent. The main
aim for thepreparation of solvents is that it has to dissolve the analytes up to
the concentration suitable for the detection.
Role of Temperature:
While temperature is a variable that can affect selectivity, a, its effect is
relatively small. Also, the k ‘ generally decreases with an increase in
temperature for neutral compounds but less dramatically for partially ionized
analytes. Snyder et al. (29) reported that an increase of 1 o C will decrease the
k ‘ by 1 to 2%, and both ionic and neutral samples.
Role of pH:

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pH is another factor in the resolution equation that will affect the selectivity of
the separation. In reversed-phase HPLC, sample retention increase when the
analyte is more hydrophobic
BUFFERS IN REVERSED-PHASE LIQUID CHROMATOGRAPHY
Selection of a buffered aqueous mobile phase for reversed-phase liquid
chromatography (RPC) may seem intimidating, but with an understanding of
the fundamental effect of pH on retention of ionic analytes and checking some
properties of the buffer options, a logical and reasonable choice can be derived.
METHOD DEVELOPMENT GUIDE
Fig1.1 selection of validation method

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Table No1.1: Limits of HPLC parameters

GUIDELINES FOR ANALYTICAL METHOD VALIDATION:
METHOD VALIDATION:
Method validation is the process by which it is established, through laboratory
studies, that the performance characteristics of the method meet the
requirements for its intended purpose. Typical analytical characteristics used
in method validation, commonly referred to as the “Eight steps of method of
validation”

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Fig1.2 Eight steps of method of validation

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Table1.2: Performance characteristics to be considered during the validation.

Method parameters Short description
Precision Random error of the method
Repeatability Precision measured under the best
condition possible (short period, one
analyst…)
Intermediate
Precision
Precision measure of the within-laboratory
variation due to different days, analysts,
equipments, etc.
Robustness Capacity of a method to remain unaffected
by small variations in the method
parameters as could Possibly occur during
the normal use of the method (pH, mobile
phase composition,…)
Reproducibility Precision measure determined by inter-
laboratory studies
Specificity Ability to determine the analyte in presence
of other compounds
Limit of detection Lowest sample concentration that can be
detected
Limit of
quantitation
Lowest sample concentration that can be
quantified with suitable bias and precision
Linearity Ability of the method to obtain test results
which are proportional to the concentration
in the sample
Range Concentration interval within the method
has a documented suitable performance
Stability Absence of an influence of time on the
concentration of the analyte in a sample

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Chapter 2. METHOD DEVELOPMENT
Method Development Using HPLC
In method development, best chromatographic conditions like the best
column, the best mobile phase, the detection wavelength etc. are used for
analysis of any drug. For the method development by this method some
information about the sample is very essential i.e. number of components
present in the sample, pKa values of different components, UV-Visible Spectra
of each analyte, solubility in different solvents, concentration range of each
component, nature of sample etc.
Parameters Affecting Changes in Chromatograph
The various parameters affecting the changes in chromatographic conditions
are
1. Flow rate
2. Temperature
3. pH
4. Ion pair reagent
5. Column efficiency
6. Capacity factor
7. Resolution
8. Retention time
9. Peak asymmetry
Method Validation (ICH Guidelines)
1. Accuracy,
2. Precision,
? Repeatability,
? Intermediate precision.
3. Specificity / Selectivity,
4. Limit of Detection,
5. Limit of Quantitation,
6. Linearity,

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7. Range,
8. Robustness,
9. System Suitability.

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3. DRUG PROFILE
VALBENAZINE
Valbenazine is used to treat Tardive dyskinesia in adults. Tardive dyskinesia is
a neurological disorder characterized by involuntary movements.
Table 3.1 General Information of Drug

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Chapter 4: REVIEW OF LITERATURE
Uttam Prasad Panigrahy* et. al.,
A novel approach was used to develop and validate a rapid, accurate,
precise, simple, efficient and reproducible isocratic Reversed Phase-High
Performance Liquid Chromatographic (RP-HPLC-DAD) method for the
estimation of Valbenazine in bulk and pharmaceutical dosage form.
Valbenazine were separated using Kromasil C18 column (250mm×4.6 mm,
5mm particle size), Waters Alliance e2695 HPLC system with 2998 PDA
detector and the mobile phase contained a mixture of 0.01M Ammonium
acetate (pH adjusted to 3.5 with orthophosphoric acid) and Methanol
(30:70, v/v). The flow rate was set to 1ml/min with the responses measured
at 309nm. The retention time of Valbenazine was found to be
3.733min.Linearity was established for Valbenazine in the range of 10-
125?g/ml with correlation coefficient (r2=0.999). The percentage recoveries
were between 100.3% to 100.6%. Validation parameters such as specificity,
linearity, precision, accuracy, robustness, limit of detection (LOD) and limit
of quantitation (LOQ) were evaluated for the method according to the
International Conference on Harmonization (ICH) Q2 R1 guidelines. The
developed method was successfully applied for the quantification and
hyphenated instrumental analysis.

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Chapter 5 .NEED FOR THE STUDY
Literature review indicates, no method is reported for quantification of bulk
drug and its capsule formation till date. Only few LC-MS/MS methods were
reported.

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6. AIM AND PLAN OF WORK
AIM:
To develop new HPLC method for the estimation of Valbenazine in dosage form.

PLAN OF WORK:
? Solubility determination of Valbenazine various solvents and buffers.
? Determine the absorption maxima of the drug in UV–Visible region in
different solvents/buffers and selecting the solvents for HPLC method
development.
? Optimize the mobile phase and flow rates for proper resolution and
retention times.
? Validate the developed method as per ICH guidelines.

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7. MATERIALS AND METHODS
Table7.1: Instruments used

Table7.2: Reagents used Chemicals

7.2 METHODS
Preparation of Mobile phase:
About 60 volumes of Acetonitrile, 40 volumes of Water and 0.5% Triethylamine
buffer (60:40: 0.5%) were mixed and sonicated for 15 mins for degassing and
the solution was filtered through 0.45 micron membrane filter.

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Preparation of 0.5% Triethylamine: About 0.5 ml of Triethylamine was
transferred into 100 ml volumetric flask and the volume was made up to mark
with water.

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Chapter 8: RESULTS AND DISCUSSIONS
8.1 Solubility Studies
These studies are carried out at 25 0C

Table 8.1: solubility studies.

8.2 Determination of Working Wavelength (?max)

8.2.1 Preparation of Standard solution
10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric
flask and dissolved in methanol and then make up to the mark with methanol
and prepare 10 µg /ml of solution by diluting 1ml to 10ml with methanol.

RESULTS:
The wavelength of maximum absorption (? max) of the solution of the drug in
mobile phase were scanned using UV-Visible spectrophotometer within the
wavelength region of 200–400 nm against mobile phase as blank. The
absorption curve shows characteristic absorption maxima at 264 nm for
Valbenazine (Fig.8.1), 248 nm was selected as detector wavelength for the
HPLC chromatographic method.

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Fig. 8.1: UV-VIS Spectrum of Valbenazine (264 nm)

8.3 METHOD DEVELOPMENT OF VALBENAZINE
Trial -1
Chromatographic conditions
Column : Inertsil ODS 3V (250×4.6× 5µ)
Mobile phase : Methanol: Acetonitrile : Water
Ratio : 50:10:40
Flow rate : 1.0 mL/min
Detection wavelength : 264 nm
Injection volume : 20µL
Run time : 15 min
Preparation of Standard solution
10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric
flask and dissolved in mobile phase and then make up to the mark with mobile
phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile
phase.

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Fig. 8.2: Chromatogram of Trail 1

Table 8.2: Results for Trail 1

Observation
Peak shape was not good and efficiency was not within the acceptance
criteria. So this trial was not considered.
Trial -2
Chromatographic conditions
Column : Inertsil ODS 3V (250×4.6 ×5µ)
Mobile phase : Methonol: ACN: Phosphate buffer
pH : 4.5

Ratio : 50:30:20
Flow rate : 1.0mL/min
Detection wavelength : 264 nm
Injection volume : 20 µL

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Run time : 10 min
Preparation of Standard solution
10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric
flask and dissolved in mobile phase and then make up to the mark with mobile
phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile
phase.
Fig 8.3: Chromatogram of Trail

Table 8.3: Results for Trail 2

Observation
From the above trial, it was found that tailing factor was not with the
system suitable limits. So this trial was not considered.

8.4 Optimised trial Chromatographic conditions
Column : Phenomenex C18 (250×4.6 ×5µ)
Mobile phase : Acetonitrile: Water : Triethylamine buffer (60:40:
0.5%) v/v
Flow rate : 1.0mL/min
Detection wavelength : 264nm

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Injection volume : 20µL
Run time : 5min
Preparation of Standard solution
10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric
flask and dissolved in mobile phase and then make up to the mark with mobile
phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile
phase.
Fig 8.4: Chromatogram of Optimised trial

Table 8.4: Results for Optimised Trial

Observation:
? All the system suitability requirements were met.
? The efficiency was more than 2000 for Valbenazine
? Hence this method was optimized.

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8.5 OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR ASSAY
Table 8.5: Optimised condition

Preparation of samples for Assay
Preparation of Standard solution
10 mg of Valbenazine was weighed and transferred in to 100 ml volumetric
flask and dissolved in mobile phase and then make up to the mark with mobile
phase and prepare 10 µg /ml of solution by diluting 1ml to 10ml with mobile
phase.
Preparation of Sample solution
Sample name: Ingrezza 10 mg capsules
Weigh 20 capsules by removing the shell then crush with mortar and pestle
then weigh a quantity of powder equivalent to 10mg of Valbenazine and
transferred in to 100 ml volumetric flask and dissolved in mobile phase and
then make up to the mark with mobile phase and prepare 10 µg /ml of solution
by diluting 1ml to 10ml with mobile phase.

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Where,
AS: Average peak area due to standard preparation
AT: Peak area due to assay preparation
WS: Weight of Valbenazine in mg
WT: Weight of sample in assay preparation
DT: Dilution of assay preparation
DS: Dilution of standard preparation
P: Purity of Valbenazine
AV: Average weight of tablets in mg
LC: Labelled claim of Valbenazine in capsules

Fig. 8.5: Chromatogram of Assay Standard-01

Fig. 8.6: Chromatogram of Assay Standard-02

Fig. 8.7: Chromatogram of Assay Standard-03

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Fig. 8.8: Chromatogram of Assay Standard-04

Fig. 8.9: Chromatogram of Assay Standard-05

Fig. 8.10: Chromatogram of Assay Sample-01

Fig. 8.11: Chromatogram of Assay Sample-02

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Fig. 8.12: Chromatogram of Assay Sample-03
Fig. 8.13: Chromatogram of Assay Sample-04

Fig. 8.14: Chromatogram of Assay Sample-05

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Table 8.6: Results for Valbenazine

Table 8.7: Results of assay

Observation
The amount of Valbenazine present in the taken dosage form was found to be
99.35 %.

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Chapter 9: VALIDATION
9.1 System Suitability& System precision
To verify that the analytical system is working properly and can give
accurate and precise results were evaluated by 10µg/mL of Valbenazine
was injected six times and the chromatograms were recorded for the same.
Table 9.1: Result for system suitability.

Acceptance criteria
1. The % RSD for the retention time of valbenazine Peaks from 6 replicate
injections of each Standard solution should be not more than 2.0
2. The % RSD for the peak area responses of valbenazine peak from 6 replicate
injections of each standard solution should be not more than 2.0%.
3. The number of theoretical plates (N) for the valbenazine peaks is not less
than 2000.
4. The Tailing factor (TP) for the valbenazine peak is not more than 2.0.
Result
The plate count and tailing factor results were found to be within the limits
and the % RSD was found to be 0.1 so system is suitable and giving precise
results.

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9.2 Method precision
Method precision was determined by injecting sample solutions of
concentration valbenazine (10?g/mL) for six timesare prepared separately.
Fig. 9.1: Chromatogram of Method Precision-01

Fig. 9.2: Chromatogram of Method Precision-02

Fig. 9.3: Chromatogram of Method Precision-03

Fig. 9.4: Chromatogram of Method Precision-04

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Fig. 9.5: Chromatogram of Method Precision-05

Fig 9.6: Chromatogram of Method Precision-06

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Table 9.2: Method precision results

Result
The % RSD of Assay for 6 Samples determinations of valbenazine found
to be within the acceptance criteria (less than 2.0%). Hence method is precise.

9.3 Linearity and range
Preparation of standard stock solution
Standard stock solutions of Valbenazine were prepared by dissolving
100 mg of Valbenazine in 100 mL of mobile phase. After that filtered the
solution using 0.45-micron syringe filter and Sonicated for 5 min further
dilutions were given in the Table 9.3.

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Table 9.3: Linearity Preparations.

Fig 9.7: Chromatogram of linearity for preparation 1.

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Fig 9.8: Chromatogram of linearity for preparation 2

Fig 9.9: Chromatogram of linearity for preparation 3.

Fig 9.10: Chromatogram of linearity for preparation 4.

Fig 9.11: Chromatogram of linearity for preparation 5.

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A graph was plotted for valbenazine against the concentrations of the
solutions and the peak areas (Table 9.9). The correlation coefficient R2 was
determined and was found to be 0.999 for valbenazine (Fig. 9.12)

Table 9.4: Linearity data.

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Fig 9.12: Graph for Linearity data.

Table 9.5: Linearity results.

Acceptance criteria
The relationship between the concentration of valbenazine and area of
valbenazine should be linear in the specified range and the correlation should
not be less than 0.99.
Result
The correlation coefficient for linear curve obtained between
concentration vs. Area for standard preparation was found to be 0.999
9.4 Specificity:
Blank solution was injected and the chromatogram was recorded for the
same as given in figure below
y = 54703x – 45.96
R² = 0.999
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2
Series1
Linear (Series1)

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Placebo solution was prepared and it was injected and the chromatogram
was recorded for the same as given in Fig. 9.14

Fig. 9.13: Chromatogram of Placebo
Table 9.14: Chromatogram of Blank
Result
Chromatograms of blank and placebo solutions had shown no peaks at the
retention times of Valbenazine.
It was observed that diluent or excipient peaks do not interfere with the
Valbenazine Peak.
9.5 Accuracy
Accuracy of the method was determined by Recovery studies. To the
formulation (preanalysed sample), the reference standards of the drugs
(50µg/ml, 100µg/ml and 150µg/ml ) were added at the level of 50%, 100%,
150%. The recovery studies were carried out three times and the percentage

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recovery and percentage mean recovery were calculated for drug is shown in
Table 9.6
Fig 9.15: Chromatogram of 50% recovery-1

Fig 9.16: Chromatogram of 100% recovery-1.

Fig 9.17: Chromatogram of 150% Recovery-1

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Fig 9.18: Chromatogram of 50% Recovery-2
Fig 9.19: Chromatogram of 100% Recovery-2

Fig 9.20: Chromatogram of 150% Recovery-2

Fig 9.21: Chromatogram of 50% Recovery-3

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Fig 9.22: Chromatogram of 100% Recovery-3

Fig 9.23: Chromatogram of 150% Recovery-3

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Table 9.6: Results for Recovery

Acceptance criteria
The Average % recovery of Valbenazine should lie between 98% and
102%.
Result
The percentage mean recovery of Valbenazine was found between 99.0 to 102.0

LIMIT OF DETECTION

= 3.3 * (551277)/51766
= 35.14µg/ml (Valbenazine)
Where, = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte.
Observation:
The LOD for this method was found to be 35.14µg/ml (Valbenazine)

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LIMIT OF QUANTIFICATION (LOQ)

= 10* (551277)/51766
= 106.48µg/ml (Valbenazine)
Where
= the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte.
OBSERVATION:
The LOQ for this method was found to be 106.48µg/ml (Valbenazine)

9.6 Robustness
The Robustness of the method was determined. The results obtained by
deliberate variation in method parameters are summarized below in Table 9.7
Fig. 9.24: Chromatogram of Valbenazine Robustness (0.8 ml/min)
Fig.9.25: Chromatogram of Valbenazine Robustness (1.2 ml/min)

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Fig. 9.26: Chromatogram of Temperature from 30 to 25°C.

Fig. 9.27: Chromatogram of Temperature from 30 to 35°C

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Table 9.7: Results for Robustness

Result
The tailing factor was found to be within the limits on small variation
of flow rate and wavelength.

9.7 Ruggedness
The ruggedness of the method was studied by the determining the analyst to
analyst variation by performing the Assay by two different analysts
Acceptance criteria:
The % Relative standard deviation of Assay values between two analysts
should be not more than 2.0%.
Fig. 9.28: Chromatogram of Analyst-01 standard
Fig. 9.29: Chromatogram of Analyst-01 sample

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Fig. 9.30: Chromatogram of Analyst-02 standard
Fig. 9.31: Chromatogram of Analyst-02 sample

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Table 9.8: Results for Ruggedness

Results:
From the above results % Assay and %RSD obtained acceptance criteria 2% so
method is rugged

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Chapter 9: CONCLUSION
A new precise, accurate, rapid method has been developed for the
estimation of Valbenazine pharmaceutical dosage form by HPLC.
From the above experimental results and parameters it was concluded
that, this newly developed method for the estimation Valbenazine was found to
be simple, precise, accurate and high resolution and shorter retention time
makes this method more acceptable and cost effective and it can be effectively
applied for routine analysis in research institutions, quality control department
in meant in industries, approved testing laboratories studies in near future
From results the proposed method is highly sensitive, precise and
accurate and it successfully applied for the quantification of API content in the
commercial formulations of Valbenazine Educational institutions and Quality
control laboratories.