45

CHAPTER-3

Zolmitriptan

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