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46
CHAPTER-3
Chapter-3 : Zolmitriptan
S. No. Name of the Sub- Title Page No.
3.1 Introduction 47-49
3.2 Experimental 49-59
3.3 Method validation 59-62
3.4 Result& Discussion 62-78
3.5 Conclusion 78
47
CHAPTER-3
3.1 INTRODUCTION
Zolmitriptan compound was considered for the method
development and method validation. Chemically it is described as (S)-
4-({3-[2-(dimethylamino)ethyl]-1H-indol-5-yl}methyl)-1,3-oxazolidin-2-
one. The empirical formula is C16H21N3O2. The molecular weight of
zolmitriptan is 287.16 . The chemical structure of the compound
depicted in fig.3.1. Zolmitriptan is a artificial tryptamine unoriginal
and show like a white crystalline powder that is freely soluble in
water. It is a discriminating serotonin receptor agonist of the 1B and
1D sub types drug, employed for the acute cure of migraine attack
with or exclusive of aura and cluster headaches63-65 .
Fig.3.1. Chemical structure of zolmitriptan
So many analytical methodologies be cited in analytical journals
for the quantitative willpower of Zolmitriptan and its appropriate
metabolites in plasma of human and further biological fluids 66-75. A
little number of achiral and chiral HPLC techniques were noticed in
favor of the identification and quantification of related substances in
the drug of zolmitriptan76-78.
48
A Significant quantity of analytical HPLC technique has been
described for the strength of mind of Zolmitriptan and its related
substances in bulk drugs as well as formulations. Numerous HPLC,
LC-MS and HPTLC techniques were useful in analytical magazines
throughout method enrichment, A few of the methodologies were
revealed by LC-MS for serum and blood samples.
In this Chapter, we made attention for the improvement of a new
stability representative isocratic RP-UPLC methodology for the
willpower of assay and process impurities in Zolmitriptan. The
impurity details of Zolmitriptan is shown in table.3.1. Forced
humiliation studies were conducted to find the stability representative
character of the methodology. System suitability, method precision,
detection limit (DL), Accuracy, quantification limit (QL) and linearity
were in arrangement as stated by ICH procedures 79-81.
Table 3.1: Details of Zolmitriptan process impurities
S.No Impurity structure Chemical name Molecular
weight
Impurity-1
(S)-4-(3-amino benzyl )
oxazolidin-2-one
216.24
49
Impurity-2
(S)-4-({3-[2-(dimethyl
amino)ethyl]-1H-indol-
5-yl}methyl)-oxazolidin-
2-one N-Oxide
303.36
Impurity-3
(S)-4-({3-[2-(dimethyl
amino)ethyl]-1H-indol-
5-yl}methyl)-
oxazolidin-2-one
273.33
3.2 EXPERIMENTAL
3.2.1 Materials
Zolmitriptan and its related impurities such as impurity-1, 2 and 3
were received as gift samples with characterized data from M/S
Inogent Laboratories private Ltd, Hyderabad, India. Purity was greater
than 99% for zolmitriptan and greater than 97% for its related
impurities. Acetonitrile, methanol and ammonium dihydrogen
phosphate was procured from M/S. Merck. All supplementary
aqueous solutions as well as the buffers for the eluent were acquired
with high purified water yield in house from Millipore water
refinement system.
50
3.2.2 Equipment
The employed Acquity UPLC system mainly outfitted of sample
manager, solvent manager with PDA detector. The productivity
indication was scrutinized and managed employing Empower
software. Water bath was employed for degradation experimentation.
Photo stability experimentations were performed in chamber of photo
stability. Thermal degradation stability experiments were employed in
dry hot air oven.
3.2.3 Chromatographic conditions
UPLC system outfitted by a degasser and quaternary pumps along
with auto sampler was used to 1µL of the sample onto the BEH C18,
1.7 μm column, 100 mm length ,2.1 mm i.d., which was reserved at
column high temperature at 40ºC. The isocratic eluent, is a blend of
0.01 M ammonium dihydrogen phosphate adjust pH9.5±0.02 with
dilute solution of ammonia and acetonitrile in the combination of
830:170 v/v, it was permit through a 0.22μm membrane filter and
finally degassed and sonication for some time and distributed at flow
rate of 0.3 mL/min keen on the detector.
3.2.4 Method development
3.2.4.1 Wave length Selection
The UV absorption spectra were generated for Zolmitriptan,
impurity-1,2 &3 with the help of PDA detector. Zolmitriptan and its
process impurities were initiate to have unreliable absorption of UV
maxima over a series of wavelength. Although it was introduced that
at on the substance of 225 nm, Zolmitriptan and process impurities
51
were carried into being to have superlative imaginable absorption of
UV. Consequently, 225 nm was preferred for the experiment and
quantification of Zolmitriptan and process impurities. The UV spectra
of related impurities are publicized in figure.3.2-3.5.
Fig.3.2 :Zolmitriptan UV spectrum:
Fig. 3.3: spectrum of Impurity-1
Fig. 3.4: spectrum of Impurity-2
52
Fig. 3.5: spectrum of Impurity-3
3.2.4.2 Selection of mobile phase and stationary phase
Zolmitriptan processes impurities such as impurity-1, 2 and 3
were having different functional groups. These impurities, illustrate
dissimilar affinities with eluents and stationary phase. Unusual
stationary phase(column) with dissimilar selectivity offers superior
division for method establishment. Specifically, two constraint are
preferred to get essential resolutions and symmetrical peaks and
separations for zolmitriptan and process impurities i.e., Selection of
the stationary phase and eluent .
3.2.4.3 Selection of Mobile phase
The processes impurities such as Impurity-1, 2 and 3 be co-eluted
with combination of eluents. Zolmitriptan is triptan derivative and
related substances of Zolmitriptan are having extensive series of
schisms and the partition of impurities primarily influenced by the
type of stationary phase (column). An isocratic eluent of buffer is a
mixture of 0.01M Ammonium dihydrogen phosphate in milli Q water,
adjusted to pH 9.5 with diluted solution of ammonia and acetonitrile
was taken for initial method development conditions for the partition
53
of Zolmitriptan and its processes impurities. Mobile phase was
degasified and filtered with the help of 0.22 microns millipore filter
paper.
3.2.4.4 Selection of stationary phase
Various stationary phases were made sure for the division of
Zolmitriptan such as Waters Acquity UPLC BEH C8, C18, Phenyl,
BEH Hilic and HSS T3 using by mobile phase specified.
The experimentation was started using Acquity HSS T3 100mm X 2.1
mm with 1.7 microns particle size.
Experiment-1:
The whole test niceties are as mentioned below
Column : Acquity HSS T3 100mm , 2.1 mm. with
1.7 microns particle size.
Eluent
Sample Preparation
:
:
Mix Acetonitrile and buffer in the relative
amount of 25:75(v/v).
0.5 mg/ml solution in diluent
Wavelength : 225 nm
Flow rate : 0.3 mL/ min
Temperature of column : 25°C
Diluent : Mobile phase
Mode of Elution : Isocratic
Elution time : 12 min
54
Fig.3.6: Zolmitriptan impurities blend solution
Observation: In this case, impurity-1 and Impurity-2 are co
eluting with each other and Impurity-3 peak shape also found to be
very broad. Further, it is found that zolmitriptan peak shape was not
symmetrical. Hence Acquity UPLC HSS T3 column is found to not
suitable for the separation of Impurity-1&Impurity-2.
Experiment-2:
For the better resolution and peak shape, 2nd experiment was
performed with the following circumstances:
Column : Acquity BEH Hilic 100mm x 2.1 mm with
1.7µm.
Mobile Phase : Acetonitrile and aq. Buffer in the
proportion of 75:25(v/v).
Sample conc. : 5 mg in 10 mL of diluent
Detection : 225 nm
Eluent Flow : 0.3 mL/ min
Column temperature : 25°C
Diluent
Elution mode
Runtime
:
:
:
Mobile phase
Isocratic mode
12 min
55
Fig. 3.7: Impurities blend solution
Observation: Here Impurity-1 and Impurity-2 are found to be co
eluting along with each other. Hence Acquity UPLC BEH Hilic column
is not appropriate suitable for the separation of Impurity-1&Impurity-
2. The chromatogram is shown in fig.3.7.
Experiment-3:
Again, 3rd trial was performed for better resolution and separation
of impurities with the following revised parameters:
Column : Acquity UPLC BEH Phenyl 100x 2.1 mm
1.7µm.
Eluent : Acetonitrile and Aq. buffer and in the
proportion of 75:25 v/v
Sample conc. : 0.5 mg/mL solution in diluent
Detection : 225 nm
Flow : 0.3 mL/ min
Column temperature : 25C
Diluent
Elution mode
Runtime
:
:
:
Mobile phase
Isocratic
12 min
56
Fig. 3.8: Zolmitriptan impurities blend solution chromatogram
Observation: Here also observed that Impurity-1 and Impurity-2
are co eluting with each other and impurity-3 found to elute very close
to impurity-1&2. Therefore Acquity BEH Phenyl column is not suitable
for the separation of Impurity-1& 2. Typical chromatogram as revealed
in fig.3.8.
Experiment-4:
Experimentation- 4 was proficient by captivating in view of the
above concern and alter the following parameters were recognized for
better division and resolution.
Column : Acquity BEH C8 100mm x2.1 mm 1.7µm.
Mobile Phase : Phosphate buffer plus Acetonitrile in the
percentage of 85:15 v/v.
Sample conc. : 0.5 mg/mL in diluent
Detection : 225 nm
Eluent Flow : 0.3 mL/ min
Column temperature : 25oC
Diluent
Mode of Elution
Runtime
:
:
:
Mobile phase
Isocratic
12 min
57
Fig .3.9: Zolmitriptan impurities blend solution chromatogram
Observation: From fig.3.9, it is found that Impurity-1, 2 and 3
were well divided, but Impurity-3 peak shape is not resolved
completely. So the method further needed to refine for improvement of
the Impurity-3 peak shape. Hence BEH C18 column is optional to get
symmetrical peak nature for the Zolmitriptan and impurities by
varying the column with the same constraints.
Experiment-5:
To conclude, experiment- 5 was proficient by attractive into
consideration of the above, the subsequent refined constraint
reputable for better and high resolution and severance of impurities.
Column : Acquity UPLC BEH C18, 100 mm X 2.1
mm X 1.7 micron.
Buffer preparation : 0.01M Ammonium dihydrogen phoshate
in water pH adjusted to 9.5 with dil.
ammonia solution.
Mobile phase
Sample preparation
:
:
Buffer :Acetonitrile in the percentage of
83:17 v/v.
0.5 mg/mL in diluent
Detection : 225 nm
Flow : 0.3 mL/min
58
Column temperature : 40° C
Diluent
Elution
Runtime
:
:
:
Mobile phase
Isocratic
12 min
Fig. 3.10: Typical chromatogram of Zolmitriptan impurities
blend solution
Observation: From the chromatogram shown in fig.3.10, it is
found to that all impurities are fine divided and found symmetrical
peak nature with good resolution. Hence Acquity BEH C18 stationary
phase is appropriate for the division of Zolmitriptan and its impurities.
Conclusion
Based on the above all experimentation on fixed phase, it was
originated to facilitate Impurity-1, 2, 3 and Zolmitriptan compounds
were well separated from each other in Acquity BEH C18, 100mm x
2.1 mm 1.7 microns column and same was applying for organization
of specificity and method of validation.
3.2.5 Specificity
The specificity of the UPLC methodology for Zolmitriptan was
conceded out in the occurrence of its related impurities namely
IMP
-2 -
1.1
97
IMP
-1 -
1.4
82
IMP
-3 -
1.7
97
ZO
LMIT
RIP
TA
N -
5.0
67
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
59
impurity-1,2 and 3. Stability representative nature and specificity of
the anticipated technique was conducted with the aid of forced
degradation studies for Zolmitriptan bulk drug. Standard degradation
studies were undertaken for stress circumstance of UV light at
254 nm, thermal at 105°C, acid hydrolysis in 0.1N HCl at 60°C,
hydrolysis of base conducted in 0.5N NaOH at 60°C, oxidation at
0.01% H2O2 and hydrolysis of water conducted at 60°C to examine
the capability of the developed LC method to divide Zolmitriptan from
its humiliation products. The degradation study period for thermal
was 7 days and UV light studies were 48 hours, 12 hours for the acid,
base, water hydrolysis and oxidation studies. The PDA detector was
used to evaluate the Peak purity of harassed samples of
Zolmitriptan. Quantification assay investigation was executed for
stress samples in opposition to qualified Zolmitriptan reference
standard. The assay of Zolmitriptan also calculated in the presence
of all the three impurities in test requirement level (i.e., 0.15%)
3.3 Method validation
3.3.1 Precision
The precision for assay method was estimated for zolmitriptan
test sample in opposition to a eligible primary reference standard. Six
independent assays were conducted and calculated the % RSD for
replicate assay determination.
Six different measures of Zolmitriptan (0.5 mg/mL) added with
0.15% level of related impurities namely impurity-1, 2 and 3 with
admiration to zolmitriptan analyte concentration were considered for
60
the evaluation of precision of the related substance method and the
RSD percentage of area for impurity-1, 2 and 3 were deliberate for six
replicate injections .
3.3.2 Quantification limit (QL) and Detection Limit (DL)
The quantification and detection limit were measured by using
signal-to-noise ratio of analyte peak 10:1 and 3:1, correspondingly, by
injecting a sequence of various concentrations of solutions with
known impurities. Precision experimentation was also executed at the
LOQ level by establishing different arrangements of all impurities for
six times, scheming the % RSD of the area of each impurity.
3.3.3 Linearity
Test solutions to evaluate linearity for the assay attitude were
arranged from Zolmitriptan stock solutions at dissimilar concentration
range from 50% to 150 % of analyte deliberation i.e, 50, 75, 100, 125
and 150 respectively. The concentration against peak area statistics
was derived by least-squares linear degeneration analysis.
Test solutions to evaluate linearity for the purity methodology were
arranged by reduced stock solutions to the necessary concentration.
The test solutions be organized at dissimilar concentration intensity
beginning 0.05% to 0.30 % .
3.3.4 Accuracy
Accuracy for the assay technique was examined at three different
levels and each concentration was conceded in triplicate, i.e. 50 %,
100% and 150 % with admiration to analyte test concentration. The
61
recovery percentage was measured from the Y-intercept and slope of
the calibration arc taken in the linearity study.
The accuracy of the related substances technique was examined
at six concentration levels and each concentration was executed in
triplicate, i.e. at 0.05 %, 0.10%, 0.15%, 0.2 %, 0.25 % and 0.3% of the
zolmitriptan analyte concentration (0.5 mg/mL). % Recoveries of the
interrelated impurities was considered by the Y-intercept and slope of
the calibration arc.
3.3.5 Robustness
The robustness of the made-up LC technique was appraised by
intentionally changing investigational situations and the resolution
between Zolmitriptan, impurity-1, 2 and 3 was credentials.
The flow rate for the eluent was 0.3 mL/min. The consequence of
flow rate of eluent on the resolution was deliberate at different flow
rates of 0.25 and 0.35 mL/min respectively, instead of 0.3 mL/min.
The consequence of the temperature on resolution was research at
35°C and 40°C as an alternative of 45°C. The consequence of pH on
the eluent was deliberate by changeable pH by −0.1 to + 0.1 units,
although other eluent composition was detained constant.
3.3.6 Solution stability
The stability of solution studies of Zolmitriptan assay methodology
was achieved by preparing the analyte sample solution and reference
standard in diluents and leaving together in closely capped in
suitable flasks at ambient situation for 48 hours. The similar analyte
62
solutions were performed for assay for each 6 hours gap up to the
achievement of the study era.
Stability of solution of Zolmitriptan and its suitable impurities in
the concerned substance methodology was achieved by parting spiked
analyte solutions in strongly capped flask at ambient temperature up
to 48 hours. comfortable of impurities namely impurity-1, 2 and 3
were resolute for every 6 hours gap up to the completion of the study
period.
3.3.7 Eluent (Mobile phase) stability
The steadiness of mobile phase for assay methodology was
executed by assaying the newly arranged analyte solutions versus
freshly organized reference standard solution for 6 hours gap up to
the end of the period i.e.48 hours. Eluent arranged was reserved
invariable throughout the study period. The % RSD for the assay of
Zolmitriptan was deliberated throughout eluent and solution
steadiness experimentation.
The eluent steadiness of related substances method was
accomplished for 48 hours by injecting the recently primed analyte
sample solutions for each 6 hrs gap. Stuffing of impurities namely
impurity-1, 2 and 3 were determined in the analyte sample.
3.4 Results and discussion
3.4.1. Specificity
During the forced degradation study, Zolmitriptan sample was
shown stable at Photo degradation, Thermal, water hydrolysis and
Acid hydrolysis. Zolmitriptan was despoiled into Impurity-2 in
63
oxidation condition and unknown peaks were observed in base
hydrolysis. All degraded samples are analyzed and initiated that
humiliation peaks are alienated from known impurities and
Zolmitriptan. The results from Peak purity assessment inveterate
that the Zolmitriptan peak is pure and homogenous in all the pressure
samples. There was no change in assay of zolmitriptan in the
occurrence of related substances (impurity-1,2 and 3) and its
humiliation products authenticates the stability demonstrating
strength of the methodology. The review of forced humiliation studies
is specified in the table 3. 2.
Peak purity study were conventional through PDA detector and
demonstrate that Zolmitriptan is peak is uncontaminated in all
circumstances. The degradation studies results are précis in Table
3.2.
Table 3.2: Zolmitriptan Degradation studies result
Stressed condition Time (hr) % Purity Peak Purity
Thermal humiliation 48 99.87 Pass
Photo humiliation 48 99.85 Pass
Peroxide hydrolysis 3 90.00 Pass
Acid hydrolysis 24 97.75 Pass
Base hydrolysis 8 53.13 Pass
Water hydrolysis 24 99.90 Pass
64
Fig. 3.11: Reference Chromatogram of Diluent
Fig 3.12: System suitability solution
Fig 3.13: Analyte Chromatogram
IMP
-2 -
1.1
97
IMP
-1 -
1.4
82
IMP
-3 -
1.7
97
ZO
LM
ITR
IPT
AN
- 5
.067
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
65
Fig 3.14: Chromatogram of Acid hydrolysis
Fig 3.15: Reference chromatogram of Base hydrolysis
Fig 3.16: Reference chromatogram of Oxidation sample
66
Fig 3.17: Reference chromatogram of Thermal humiliation
Fig 3.18: Reference chromatogram of Photo Degradation
3.4.2. Optimized method
From on the over study, the subsequent chromatographic
methodology was preferred for the division and quantification of
Zolmitriptan and its related substances.
The chromatography stationary phase (column) is BEH C18,100
mm, length x 2.1 mm with 1.7 µm particles. The eluent was prepared
by amalgamation buffer and acetonitrile (83: 17; v/v) and the buffer
consists of 0.01M ammonium hydrogen phosphate pH attuned to 9.5
with ammonia. The eluent flow reserved at 0.3 mL/min, column
temperature preserve at 40 °C, the nature of the peak of zolmitriptan
67
was established to be regular. In chromatographic optimized
circumstances of Zolmitriptan, impurity-1, 2 and 3 were alienated
with resolution exceeding 2, representative RT were about 5.06, 1.48,
1.19and 1.79 min, respectively (Fig3.12) . The outcome of system
suitability solution is mentioned in Table-3.03 and the developed RP-
UPLC technique was originate to be unambiguous for Zolmitriptan
and its associated impurities specifically- impurity-1, 2, and 3
(Table-3.01)
3.4.3 Method validation results for purity
3.4.3.1 System suitability
In the system suitability analyte solution was inserted into UPLC
for related substance method and system suitability constraints like
tailing factor, resolution and number of theoretical plates were
estimated (Table-3.3).
Table 3.3: System suitability results
S. No Name Rt(min) RRT Resolution,
Rs
Theoretical
plates, N
Tailing
factor, T
01 Impurity-1 1.48 0.29 --- 3604 1.1
02 Impurity-2 1.19 0.24 2.4 1317 1.2
03 Impurity-3 1.79 0.35 3.0 3627 1.2
04 Zolmitriptan 5.06 1.00 23.3 14878 1.2
68
3.4.3.2 Limit of detection and quantification
The detection limit for Impurity -1, 2 and 3 were established to be
0.013 %, 0.007 %, and 0.007 % correspondingly. The Quantification
Limit for Impurity -1, 2 and 3 were established to be 0.04 % , 0.022 %
and 0.02 % correspondingly. The final outcomes are recapitulate in
the underneath table.
Table 3.4: Results of Detection Limit and quantification Limit
Conc. Impurity -1 Impurity -2 Impurity -3
(%) LOD 0.013 0.007 0.007
(%) LOQ 0.04 0.022 0.02
3.4.3.3 Precision and accuracy at quantification limit level
The impurities percentage RSD at the LOQ level for Impurity-1, 2
and 3 were 1.2%, 1.5% and 5.0 %. The percentage recovery at LOQ
level for Impurity-1, 2 and 3 were 99.8, 99.9 and 99.5
correspondingly. The outcome are précis in the beneath table.
Table 3.5: Accuracy at quantification level and Precision results
S. No Name of the Impurity % RSD (n=6) % Recovery (n=3)
1 Impurity-1 1.2 99.8
2 Impurity-2 1.5 99.9
3 Impurity-3 5.0 99.5
3.4.3.4 Linearity
The Linear calibration plots for the processed impurities described
as Imp-1, 2 and 3 were found over the calibration vary experienced,
i.e. The achieve concentration levels 0.05% to 0.3%. The correlation
69
coefficient be beyond 0.999. The outcome illustrate that an
outstanding relationship present between the concentration and the
peak area of imp-1, 2 and 3. The linearity computation were prepared
right through Microsoft XL data computations.
Table 3.6: Impurity-1 Linearity data
S. No Concentration
(%)
Area of Impurity-1
1 0.05 2088
2 0.10 4175
3 0.15 6217
4 0.20 8395
5 0.25 10445
6 0.30 12685
Correlation coefficient 0.99992
Slope 42271.71
Y-Intercept -63.4667
Fig 3.19: Zolmitriptan Impurity-1 Linearity graph
70
Table 3.7 Linearity data Impurity-2
S. No Concentration (%) Area of Impurity-2
1 0.05 5606
2 0.10 11212
3 0.15 16959
4 0.20 22847
5 0.25 29530
6 0.30 34338
Correlation coefficient 0.99943
Slope 116858.3
Y-Intercept -368.2
Fig. 3.20 Zolmitriptan Impurity-2 graph
71
Table 3.8 Zolmitriptan Impurity-3 Linearity data
S. No Concentration (%) Area of Impurity-3
1 0.050 6072
3 0.100 12144
5 0.150 17695
6 0.200 24697
7 0.250 30661
8 0.300 36655
Correlation coefficient 0.99968
Slope 123124.6
Y-Intercept -226.1
Fig 3.21: Zolmitriptan Impurity-3 graph
3.4.3.5 Accuracy:
The recovery % of all associated impurities in Zolmitriptan analyte
is exposed in below Table 3.9
72
Table 3.9: Accuracy in % Recovery:
Concentration(%) Imp-1(%)
(n=3)
Imp-2(%)
(n=3)
Imp-3(%)
(n=3)
0.05% 98.9 99.0 99.3
0.10% 97.7 97.78 99.1
0.15% 99.2 100.8 99.8
0.20% 99.5 99.8 96.7
0.25% 101.6 99.5 98.9
0.30% 96.8 99.5 100.5
3.4.3.6 Method Precision:
In the purity method, method precision was verified by
introducing six individual measures of Zolmitriptan impaled by 0.10%
of Impurity-1, 2 and 3. The % R.S.D of the area for each of Impurity-
1, Impurity-2 and Impurity-3 were considered. The outcome were
recapitulated in the underneath table
Table 3.10: Results of method Precision data
S. No Preparation Imp-1 area Imp-2
area
Imp-3
area
1 1 6826 13550 12061
2 2 6857 13670 12046
3 3 6860 13333 12069
4 4 6994 13274 12146
5 5 7002 13403 12034
6 6 7060 13798 12403
Average 6933.2 13504.7 12126.5
Standard Deviation 97.123 204.18 141.043
%RSD 1.40 1.51 1.16
73
3.4.3.7 Robustness:
The Robustness of the methods was fruitfully demonstrated and in
all the intentionally dissimilar chromatographic circumstances (flow
rate of eluent, pH of the eluent and temperature ), the resolution
between significant pair, i.e., Impurity-2 and 3 was above 2.0,
demonstrated the forcefulness of the methodology. The detailed
experimentation data is depicted in Table 3.11.
Table.3.11: Results for robustness data
Parameter
Relative retention time ( RRT, in minutes)
Imp -1 Imp –2 Imp –3 Tailing
factor(T)
Theoretical
plates(N)
Resolution
(Rs)
1. Flow Rate , mL/min (± 0.05 ml/min)
0.25 0.28 0.24 0.37 1.2 13282 2.2
0.30 0.29 0.24 0.35 1.2 14878 2.4
0.35 0.28 0.24 0.37 1.2 12084 2.2
2. Temperature((± 5°C)
35°C 0.28 0.23 0.36 1.3 12363 2.4
40°C 0.29 0.24 0.35 1.2 14878 2.4
45°C 0.31 0.25 0.38 1.3 11614 2.2
3. pH (± 0.1 of the set pH)
9.4 0.28 0.23 0.37 1.2 12384 2.2
9.5 0.29 0.24 0.35 1.2 14878 2.4
9.6 0.28 0.23 0.38 1.2 12804 2.2
74
3.4.3.8 Solution and Eluent(Mobile phase) stability
No considerable difference were identified in the substance of three
impurities 1,2&3, and other degradation impurities are also not
noticed for the period of the solution and eluent stabilities tests when
examined using the related substance methodology. The eluent and
solution steadiness trial information authenticate that the analyte
solutions and eluent employed throughout the quantitative
determination of impurities were stable for as a minimum 48 hours.
The outcomes are précis in the beneath table 3.12.
Table 3.12: Results of Solution stability data
Duration Imp-1
(%)
Imp-2
(%)
Imp-3
(%)
Any other
impurity(%)
Purity
(%)
SS Initial 0.01 ND 0.01 0.01 99.96
After 12 hrs 0.01 ND 0.01 0.01 99.96
After 24 hrs 0.01 ND 0.01 0.01 99.96
After 48 hrs 0.01 ND 0.01 0.01 99.96
Table 3.13: Mobile phase stability data
Duration Imp-1
(%)
Imp-2
(%)
Imp-3
(%)
Any other
imp(%)
Purity
(%)
SS Initial 0.01 ND 0.01 0.01 99.96
After 12 hrs 0.01 ND 0.01 0.01 99.96
After 24 hrs 0.01 ND 0.01 0.01 99.96
After 48 hrs 0.01 ND 0.01 0.01 99.96
75
3.4.4 Method validation results for Assay
3.4.4.1 System suitability
The system suitability of analyte sample was injected into UPLC
for quantitative estimation and evaluated the plate count, Tailing
factor and Relative standard deviation for replicate injections
Table 3.14:Zolmitriptan assay System suitability Results
S. No. Parameter Observed result
1 %RSD 0.17
2 Tailing factor 1.2
3 Theoretical plates 14689
3.4.4.2 Linearity
The calibration arc for linearity of the assay methodology was
acquired in excess of the calibration series tested, i.e. 125 –
375 μg/mL and correlation coefficient acquired was found exceeding
0.999.
Table 3.15: Zolmitriptan Linearity results
S. No Level (%) Area of Zolmitriptan
1 50 8565412
2 75 12848094
3 100 17130728
4 125 21413498
5 150 25696380
Correlation coefficient ( r2 ) 0.999996
Slope 17131494.4
Y-Intercept -431.2
76
Fig 3.22: Zolmitriptan Linearity graph
3.4.4.3 Precision
The % RSD for precision experimentation was within 0.25% in
assay of Zolmitriptan throughout the analysis.
Table 3.16: Zolmitriptan precision results
3.4.4.4 Accuracy
The percentage recovery for assay of Zolmitriptan in bulk drug
sample was range starting 99.1 to 100.7%.
Preparation Zolmitriptan Assay(%w/w)
1 99.8
2 99.7
3 99.4
4 99.2
5 99.3
6 99.7
Average 99.52
STDEV 0.248
%RSD 0.25
77
Table 3.17: Zolmitriptan Accuracy data
3.4.4.5 Robustness
The relative standard deviation for six replicate injection from
system suitability solution found below 0.5%. Results mentioned in
below table 3.19.
Table 3.18: Robustness data
System
suitability
parameters
Parameters and Results
Flow Rate Variation Temperature
Variation pH Variation
As
such
0.25
mL/min
0.35
mL/min
At 35°C At 45°C pH at 9.4 pH at
9.6
% RSD 0.17 0.42 0.37 0.32 0.19 0.21 0.27
3.4.4.6 Stability of solution and Mobile phase
There is no change experiential in assay during the solution
stability and eluent stability throughout the time period. Results
mentioned below in table 3.19.
Concentration (mg/ml) % Recovery (n=3)
0.05 99.1
0.075 99.7
0.10 99.1
0.125 99.6
0.15 100.7
78
Table 3.19 Zolmitriptan Stability of Solution and Mobile phase
3.5 Conclusion
The simple RP-UPLC isocratic technique developed for
quantification of Zolmitriptan and processed impurities is specific,
accurate, precise and rapid. The established technique was completely
authenticated based on adequate data throughout the method of
validation factors tested. The optimized technique can be employed for
the analysis of regular production drugs as well as stability samples.
Interval Solution stability
% Assay
Mobile phase stability
% Assay
Initial 99.7 99.7
After 6 hrs 99.5 99.5
After 12 hrs 99.4 99.8
After 24 hrs 99.3 99.5
After 48 hrs 99.6 99.3