Journal of ISSN: 2473-0831 JAPLR

Analytical & Pharmaceutical Research
Research Article
Volume 4 Issue 1 - 2017
Development and Validation of RP-HPLC Method for the Identification of Process Related Impurities of Zolmitriptan
Nachiket S Dighe1*, Sachin Kisan Hodgar1 and Deepak S Musmade2
1Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, India
2Department of Pharmaceutical Chemistry, Sanjivani College of Pharmaceutical Education & Research, India
Received: January 31, 2017 | Published: February 06, 2017
*Corresponding author: Nachiket S Dighe, Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, A/P- Loni Bk. Taluka -Rahata, Ahmednagar 413736, India, Tel: 09890215729; Email:
Citation: Dighe NS, Hodgar SK, Musmade DS (2017) Development and Validation of RP-HPLC Method for the Identification of Process Related Impurities of Zolmitriptan. J Anal Pharm Res 4(1): 00094. DOI: 10.15406/japlr.2017.04.00094

Abstract

The study was focused toward synthesis, characterization and quantification of 3-Ethyl-indole impurity in Zolmitriptan formulations by Reverse Phase High Performance Liquid Chromatography method. The synthesis of a process related impurity of Zolmitriptan was successfully carried out by Fischer indole procedure. The impurity was purified by column chromatography. Characterization was done by I.R, 1H-NMR, 13C-NMR and GC-MS. Based on the spectral data, the structure of impurity was characterized as 3-Ethyl-indole. An efficient isocratic RP-HPLC was developed and validated according to ICH guidelines with respect to specificity, accuracy, linearity and precision. The validated HPLC method was used for detection and quantitation of 3-Ethyl-indole, a process related impurity of Zolmitriptan, from Zolmitriptan tablet formulations. The above method was found to be specific, accurate, precise, rugged and robust and can be used for routine analysis.

Keywords: Zolmitriptan; Fischer indole; Column chromatography; iIsocratic; Rugged; Robust; Specificity; Accuracy; Linearity; Precision

Abbreviations

NMR: Nuclear Magnetic Resonance; HPLC: High Performance Liquid Chromatography; RP-HPLC: Reverse Phase High Performance Liquid Chromatography; HPTLC: High Performance Thin Liquid Chromatography; TLC: Thin Liquid Chromatography; API’s: Active Pharmaceutical Ingredient’s; LOD: Limit of Detection; LOQ: Limit of Quantitation; FTIR: Fourier Transform Infra Red spectroscopy

Introduction

In Pharmaceutical World, an impurity is considered as any other organic materials, besides the drug substances, or ingredients, arises out of synthesis or unwanted chemicals that remains with Active Pharmaceutical Ingredient’s (API’s). The impurity may be developed either during formulation or upon aging of both API’s and formulations. Presence of impurities in trace quantity in drug substance or drug product is inevitable. Therefore, their level should be controlled and monitored. They reinforce or diminish the pharmacological efficacy of the Active Pharmaceutical Ingredient’s [1].

ICH defines impurities profile of a drug materials is “A description of the identified and unidentified impurities, present in a new drug substance.” For Pharmaceutical products, impurities are defined as “substance in the product that are not the API itself or the excipient used to manufacture it ” i.e. impurities are unwanted chemical that remains within the formulation or API in small amounts which can influence Quality, Safety and Efficacy, thereby causing serious health hazards [2].

Qualification of the impurities is the process of acquiring and evaluating data that establishes biological safety of an individual impurity; thus, revealing the need and scope of impurity profiling of drugs in pharmaceutical research. Identification of impurities is done by a variety of Chromatographic and Spectroscopic techniques, either alone or in combination with other techniques [3-5]. There are different methods for detecting and characterizing impurities with TLC, HPTLC, and HPLC etc. Conventional Liquid Chromatography, particularly, HPLC has been exploited widely in field of impurity profiling; the wide range of detectors, and stationary phases along with its sensitivity and cost effective separation have attributed to its varied applications. Various regulatory authorities like ICH, USFDA, Canadian Drug and Health Agency are emphasizing on the purity requirements and the identification of impurities in Active Pharmaceutical Ingredient’s (API’s) [6-8]. According to ICH guidelines on impurities in new drug products, identification of impurities below the 0.1% level is not considered to be necessary, unless potential impurities are expected to be unusually potent or toxic. According to ICH, the maximum daily dose qualification threshold is considered as follows; ≤ 2g/day 0.1% or 1 mg per day intake (whichever is lower) ≥ 2g/day 0.05% [9-12].

Materials and Methods

Materials reagents and chemicals

Butanaldehyde, silica gel, hydrochloric acid etc. were purchased from Merck Chemicals Pvt. Ltd. Nasik, MS, India are of AR grade. Methanol, benzene of AR grade and the acetonitrile, methanol and water of HPLC grade were purchased from Merck Chemicals Pvt. Ltd. Nasik, MS, India. The Zolmitriptan tablet formulations of different batches were purchased from local market of Kopargaon.

 Melting points were determined by open capillary method and are uncorrected. The NMR spectra were recorded on sophisticated multinuclear FT-NMR Spectrometer model Advance-II (Bruker) using dimethylsulfoxide-d6 as solvent and tetramethylsilane as internal standard for 1H and 13C NMR. IR spectra were recorded on Shimadzu FTIR-8400S spectrophotometer using KBr disc method.

Chromatographic conditions

The quantitation of indole from formulation was carried out by HPLC method. The LC20AD Prominence Liquid Chromatography SPD20-A Shimadzu, Japan with UV-Vis detector and C18 column with dimension on 25 x 0.6cm was used for the method development with flow rate 1.0ml/min at wavelength 236nm. The methanol: acetonitrile: water in proportion of (35v:38v:27v) as a mobile phase, for development of chromatogram. The method was validation for synthesized compound and various parameters according to ICH guidelines (Q2B) were studied.

General method for 3-ethyl-indole synthesis

Synthesis was started by Fischer indole synthesis using phenyl hydrazine with appropriate aldehyde and hydrochloric acid was refluxed in presence of methanol for 2hrs and then filtered to offer indole derivative [13].

Preparation of standard solution

100ppm (100µg/ml) of standard solution of synthesized compound was prepared by dissolving in methanol. 10mg of standard synthesized compound was dissolved in methanol up to 100ml. From this stock, 1ml of solution was pipette out and diluted by the solvent up to 10ml to prepare 10ppm (10µg/ml) of solution

Preparation of test solution

Test solution of formulation with different concentration from 1ppm to 10ppm was prepared using same solvent. Validation parameters were carried out for impurity by calculating range, linearity, accuracy, precision, ruggedness, robustness, LOD and LOQ as per ICH guidelines.

Analytical method validation

A suitable analytical method was developed and validated for identification. New drug development requires meaningful and reliable analytical data to be produced at various stages of development.

Preparation of mobile phase

The selection of mobile phase was according to polarity and non-polarity of solvents. The methanol: acetonitrile: water was selected as mobile phase in ratio of 35:38:27 and was filtered on membrane filter (0.45μ) to remove degassing and were stirred for 10-15min.

Preparation of stock solution standard

The stock solution was prepared according to the standard procedure viz., 10mg of synthesized compound was accurately weighed on analytical balance and using mobile phase it was dissolved to make volume up to 100 ml stock solution. The sample was prepared in the ppm in the range of 1-6ppm in concentrations respectively for the method validation by HPLC.

Preparation of sample solution (formulation)

Stock solutions of 2 different batches of Zolmitriptan marketed formulation of 100ppm in 100ml volumetric flask were prepared. For the tablet formulation 20tablets from each 2tablet batch were crushed respectively. The powder of this formulation equivalent to 10 mg of the drug was used to prepare the stock solution. Further dilute to 1ppm, 2ppm, and so on, were prepared by taking 0.1ml, 0.2ml and so on of standard test solution and diluting it to 10ml. Validation experiment was performed to demonstrate system suitability, linearity, precision, accuracy study, ruggedness and robustness as per ICH guidelines.

Results and Discussion

Physicochemical properties (Table 1).

S. No

Characteristics

Impurity

4

Molecular formula

C10H11N

5

Molecular weight

145

6

Melting point

138°C

7

Elemental Analysis

C

82.72%

H

7.64%

N

9.65%

Table 1: Physicochemical properties of synthesized compound.

Thin layer chromatography

The sample preparation was done by dissolving solute in Methanol.

The Mobile phase Benzene: Methanol (6:1 v/v) Rf value- 0.80 (Figure 1).

Figure 1: TLC of Synthesized compound.

R f = 3.8 4.7 R f =0.80 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGsb qcfa4aaSbaaeaajugWaiaadAgaaKqbagqaaKqzGeGaeyypa0tcfa4a aSaaaOqaaKqzGeGaaG4maiaac6cacaaI4aaakeaajugibiaaisdaca GGUaGaaG4naaaacqGHsislcaWGsbqcfa4aaSbaaeaajugWaiaadAga aKqbagqaaKqzGeGaeyypa0JaaGimaiaac6cacaaI4aGaaGimaaaa@4C10@

Characterization of synthesized compound

The synthesized compound was characterized by using Advance Analytical Techniques such as UV

spectra, I.R spectra, 1H-NMR, 13C-NMR and GC-MS. Following are the recorded spectra.

UV- Spectra

The λmax of 3-ethyl-indole in methanol as solvent was found at 236nm. Since the compound substitute benzene chromophore, λmax confirms the structure (Figure 2).

Figure 2: UV spectra of synthesized compound.

I.R Spectra (Figure 3).

Figure 3: IR spectra of impurity 3-ethyl-indole.

Spectral data: The major functional group are primary amine, nitro, and carbonyl group obtained peak in IR spectra are as follows.

IR (KBr) cm-1

3341 (NH- stretching), 2850 (aliphatic –CH stretching), 2921, 2853 (aromatic C-H stretching), 1368 (C-H bending), 1297 (CH3 bending), 744,750,766 (benzene ring bending). The spectral data confirm the structure of compound.

NMR- Spectra

1H NMR: For recording the 1H NMR spectra of 3-ethyl-indole, the synthesized compound was dissolved in Deuteriated Dimethyl Sulphoxide and the spectrum was recorded (Table 2).

S. No

Group of Protons

Chemical shift δ (ppm)

a.

Proton attached to CH2

2.27

b.

Proton attached to CH3

3.3

c.

Alkene proton of benzene

6.4-6.8

d.

Proton of NH

5

e.

Proton of –CH of pyrrole

6.2

Table 2: 1H- NMR Spectra data.

The PMR data supports the structural confirmation of synthesized compound 3-ethyl-indole.

GC-MS

The Q-TOF Micro mass (YA-105) spectrometer capable of recording High Resolution Spectrum (HRMS) both in Atomic Pressure Chemical Ionization (APCI) and Electron Spray Ionization (ESR) were used for quantitation of synthesized impurities. The m/e ratio was calculated for synthesized compound. m/e for the synthesized compound was found at 145 confirms the mass of the synthesized impurity.

UV method validation of impurity

UV Method was developed for synthesized compound was carried out as per ICH (Q2B) guidelines.

Range: The range was found to be 1-18µg/ml.

Linearity: Calibration curve data was constructed in the range of 1 to 18µg/ml. Beer’s law was observed over this concentration range. The correlation coefficient (R2) was found to be 0.995. The regression equation Y= 0.054x+ 0.009 was found to be linear (Table 3 & Figure 4).

S. No

Concentration (ppm)

Absorbance at 236 nm

1

2

0.16

2

4

0.217

3

6

0.324

4

8

0.442

5

10

0.565

6

12

0.629

7

14

0.756

8

16

0.912

9

18

1.007

10

20

1.119

Table 3: Linearity of impurity by UV.

Figure 4: Graph of Linearity of synthesized compound by UV.

Precision (Table 4).

S. No

Concentration (ppm)

Absorbance at 236nm

Mean

S.D

% RSD

1

6

0.315

0.319

0.0024

0.8777

2

6

0.321

3

6

0.324

4

6

0.319

5

6

0.321

6

6

0.318

7

6

0.319

Table 4: Precision by UV.

Intraday precision (Table 5).

S. No

Conc. (ppm)

Abs. At 236nm after 4hr. Reading

Mean

SD

%RSD

1

6

0.309

0.316

0.0035

1.1071

2

6

0.318

3

6

0.32

4

6

0.318

5

6

0.317

6

6

0.315

7

6

0.316

Table 5: Intraday precision after 4 hour.

Interday precision: (Table 6).

S. No

Conc. (ppm)

Abs. At 236 nm after 24hr. Reading

Mean

S.D

%RSD

1

6

0.308

0.314

0.0032

1.1012

2

6

0.317

3

6

0.318

4

6

0.316

5

6

0.315

6

6

0.314

7

6

0.314

Table 6: Interday precision after 24 hours.

In the result of intraday and interday precision expressed by %RSD. Since variation in %RSD was not much, state the preciseness of the method.

Robustness (Table 7).

S. No

Conc. (ppm)

Abs. at 236nm (Single I)

Abs. at 236 nm (Double II)

Mean

S.D

%RSD

I

II

I

II

I

II

1

6

0.313

0.315

0.316

0.319

0.0031

0.0028

0.981

0.8771

2

6

0.317

0.321

3

6

0.323

0.324

4

6

0.316

0.319

5

6

0.318

0.321

6

6

0.315

0.318

7

6

0.315

0.319

Table 7: Results of robustness study by change in Instrument.

Robustness of the method was determined by using different instrument and the respective absorbance was noted and the result was indicated by %RSD. The method was found to be robust.

Ruggedness (Table 8 & 9).

S. No

Conc. (ppm)

Absorbance

Mean

S.D

%RSD

Analyst I

Analyst II

I

II

I

II

I

II

1

6

0.315

0.312

0.319

0.317

0.0028

0.0026

0.8777

0.8201

2

6

0.321

0.318

3

6

0.324

0.32

4

6

0.319

0.318

5

6

0.321

0.317

6

6

0.318

0.32

7

6

0.319

0.317

Table 8: Result of ruggedness study by change in Analyst I and II.

The Ruggedness was carried out by change in analyst and the difference of %RSD is negligible indicates that the method is rugged.

S. No

Parameter

SD

%RSD

1

Precision

0.0028

0.8777

2

Intraday precision

0.0035

1.1071

3

Interday precision

0.0032

1.1012

4

Robustness

0.0029

0.9293

5

Ruggedness

0.0027

0.8489

Table 9: Summary of Precision.

The summary of the precision is given in the above table and % RSD was found to be ≤ 2.

Accuracy: The accuracy with known concentration of synthesized compound in formulation was determined. The recovery assessment was performed by the analysis of Zolmitriptan formulation spiked with known amount of impurity at three concentration level in triplicate of 50%. 100%, 150% and was found to be accurate as shown in above Table 10 & 11.

S. No

Drug/ Formulation

Percentage Recovery

Mean

S.D.

%RSD

50%

100%

150%

1

Tablet I

97.61

98.44

98.67

98.24

0.5575

0.5674

2

Tablet II

98.19

98.85

98.39

98.47

0.3384

0.3439

Table 10: Result of recovery study by UV.

S. No.

Drug / Formulation

Amount of Drug (µg/ml)

Amount of Impurity Added (µg/ml)

Amount Recovered(µg/ml)

1

Tablet I

10

5

14.63

10

10

19.68

10

15

24.66

2

Tablet 2

10

5

14.72

10

10

19.77

10

15

24.54

Table 11: Recovered amount of impurity by UV.

Limit of detection:

LOD= 3.3×Standard deviation Slope MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIZaGaaiOl aiaaiodacqGHxdaTqaaaaaaaaaWdbiaadofacaWG0bGaamyyaiaad6 gacaWGKbGaamyyaiaadkhacaWGKbGaaeiiaiaadsgacaWGLbGaamOD aiaadMgacaWGHbGaamiDaiaadMgacaWGVbGaamOBaaGcpaqaaKqzGe Gaam4uaiaadYgacaWGVbGaamiCaiaadwgaaaaaaa@5543@

LOD= 3.3×0.002819 0.054 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIZaGaaiOl aiaaiodacqGHxdaTcaaIWaGaaiOlaiaaicdacaaIWaGaaGOmaiaaiI dacaaIXaGaaGyoaaGcbaqcLbsacaaIWaGaaiOlaiaaicdacaaI1aGa aGinaaaaaaa@498B@

LOD=0.1722 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpcaaIWaGaaiOlaiaaigdacaaI3aGaaGOm aiaaikdaaaa@3E59@

Limit of quantitation:

LOQ= 10×Standard deviation Slope MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIXaGaaGim aiabgEna0cbaaaaaaaaapeGaam4uaiaadshacaWGHbGaamOBaiaads gacaWGHbGaamOCaiaadsgacaqGGaGaamizaiaadwgacaWG2bGaamyA aiaadggacaWG0bGaamyAaiaad+gacaWGUbaak8aabaqcLbsacaWGtb GaamiBaiaad+gacaWGWbGaamyzaaaaaaa@5499@

LOQ= 10×0.002819 0.054 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIXaGaaGim aiabgEna0kaaicdacaGGUaGaaGimaiaaicdacaaIYaGaaGioaiaaig dacaaI5aaakeaajugibiaaicdacaGGUaGaaGimaiaaiwdacaaI0aaa aaaa@48E1@

LOQ=0.5220 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpcaaIWaGaaiOlaiaaiwdacaaIYaGaaGOm aiaaicdaaaa@3E63@

The LOD and LOQ were found to be 172.2ng and 522.0ng respectively, which indicate the sensitivity of method (Table 12).

S. No

Parameter

Observation

1

Linearity range

1-18 μg/ml

2

Slope

0.0549

3

Intercept

0.0095

4

Correlation coefficient

0.995

5

LOD

0.17227

6

LOQ

0.52203

Table 12: Summary of method validation.

The developed UV method was validated as per ICH guidelines and the method is simple, cost effective to determine 3-ethyl-indole impurity in tablet formulation of Zolmitriptan for routine analysis.

HPLC method validation

The ICH Q2B guidelines discuss the analytical method validation on HPLC. Currently the vast majority of process-related impurity determinations are performed by HPLC. It offered the desired sensitivity for trace level determinations with a high degree of automation. A wide variety of stationary phases and operation modes make HPLC applicable to all drug classes. The typical detection limits for process-related impurities by HPLC are 0.1% or lower and this can be routinely met in the majority of circumstances using conventional UV detectors. These methods involved the prediction of likely impurities within the synthetic process, their isolation and identification by suitable analytical techniques. The last step of the present study was to develop, validated HPLC method for detection and quantification of 3-ethyl-indole impurity in tablet formulations.

HPLC chromatograph of zolmitriptan (Figure 5 & Table 13).

Figure 5: HPLC Chromatogram of Zolmitriptan.

Reten. Time [min]

Area [mV.s]

Height [mV]

Area [%]

Height [%]

WO5 [min]

1

1.46

2.424

0.289

0.1

0.3

0.13

2

1.85

11.705

1.278

0.7

1.1

0.11

3

4.063

37.267

3.919

2.2

3.4

0.15

4

4.627

25.732

2.608

1.5

2.3

0.17

5

4.817

52.839

5.63

3.1

4.9

0.14

6

9.587

1568.718

100.231

92.3

88

0.23

Total

1698.684

113.955

100

100

Table 13: The Retention time of Zolmitriptan was 9.5 min.

HPLC chromatogram of synthesized compound (Figure 6 & Table 14).

Figure 6: HPLC chromatogram of synthesized compound.

The retention time of impurity was 10.5 min and it shows a single peak which indicates purity of compound.

Reten. Time [min]

Area [mV.s]

Height [mV]

Area [%]

Height [%]

WO5 [min]

1

1.863

9.288

0.859

5.6

8

0.15

2

4.65

13.445

0.993

8.1

9.3

0.21

3

8.223

8.412

0.653

5.1

6.1

0.19

4

10.597

135.253

8.174

81.3

76.5

0.25

Total

166.397

10.678

100

100

Table 14: HPLC chromatogram of synthesized compound.

The retention time of impurity was 10.5 min and it shows a single peak which indicates purity of compound.

HPLC chromatogram of tablet (Figure 7 & Table 15).

Figure 7: HPLC Chromatogram of Zolmitriptan Tablet.

The retention time of Zolmitriptan tablet was found at 9.6 min.

Reten. Time [min]

Area [mV.s]

Height [mV]

Area [%]

Height [%]

WO5 [min]

1

1.87

19.233

2.024

10.7

16.1

0.12

2

4.643

39.227

2.71

21.9

21.6

0.22

3

9.687

120.869

7.84

67.4

62.4

0.23

Total

179.33

12.573

100

100

Table 15: HPLC Chromatogram of Zolmitriptan Tablet.

The retention time of Zolmitriptan tablet was found at 9.6 min.

Optimized chromatographic condition (Table 16).

Chromatographic Conditions

Shimadzu HPLC System

Mobile phase

Methanol: Acetonitrile: Water (35:38:27)

Column

ARP-C18 (250 mm X 4.6 mm), 5μ column

Flow rate

1ml/min

Wavelength detection

236nm

Injection volume

20μl

Temperature

Ambient

Retention time

10.5min

Run time

15min

Table 16: Optimized chromatographic condition for RP-HPLC.

Linearity (Figure 8 & Table 17).

Figure 8: Graph of linearity of synthesized compound by HPLC.

The linearity of the proposed method was estimated by regression analysis at six concentration levels in the range of 1-6µg/ml for intermediate. The correlation coefficient (R2) was found to be 0.999 and intercept Y= 99.34x+19.42 was linear.

S. No

Concentration (ppm)

Area (mill volts) at 236nm

1

1

123.17

2

2

215.68

3

3

312.27

4

4

419.19

5

5

513.62

6

6

618.54

Table 17: Result of Linearity by HPLC (Peak area vs. Conc.).

Precision: The precision of the intermediate was quantified for repeated concentration of 4µg/ml in range and was reliable with their area of chromatogram as shown in above table. The Standard deviation (SD) and Relative standard deviation (RSD) was found to be 1.331 and 0.317 respectively (Table 18).

S. No

Concentration (ppm)

Peak area (mV) at 236nm

Mean

SD

%RSD

1

4

419.19

419.89

1.331

0.317

2

4

418.98

3

4

421.9

4

4

419.9

5

4

418.39

6

4

421.01

Table 18: Precision by HPLC.

Intraday precision after 4hours (Table 19).

S. No

Conc. (ppm)

Peak Area after 4hour at 236nm

Mean

S.D

%RSD

1

4

418.83

417

2.25

0.539

2

4

413.15

3

4

418.39

4

4

417.12

5

4

415.86

6

4

418.99

Table 19: Result of Intraday precision after 4 hours.

Interday precision after 24hours (Table 20).

S. No

Conc. (ppm)

Peak Area after 24hour at 236nm

Mean

S.D

%RSD

1

4

423.12

424.42

1.76

0.414

2

4

422.6

3

4

426.66

4

4

424.24

5

4

423.42

6

4

426.53

Table 20: Intraday precision after 24hours.

The intra and interday precision was carrying out and difference in %RSD was found not much varies and remains less than 2% indicate preciseness of method.

Robustness (Table 21).

S. No

Conc. (ppm)

Peak Area (mV) 0.8ml/min

Mean

S.D

%RSD

1

4

787.12

789.07

3.122

0.395

2

4

784.96

3

4

793.21

4

4

786.68

5

4

791.48

6

4

789.07

Table 21: Results of Robustness study by change in flow rate.
At flow rate of 0.8 ml/min

The robustness of the Intermediate was performed for change in flow rate upto 0.8 ml/min and method was robust with standard deviation 3.122 and relative standard deviation 0.395.

Ruggedness (Table 22 & 23).

S. No

Conc. (ppm)

Peak Area in mV

Mean

S.D

%RSD

Analyst I

Analyst II

I

II

I

II

I

II

1

4

419.19

418.8

419.94

420.66

1.392

1.481

0.3314

0.352

2

4

418.98

420.18

3

4

421.9

420.36

4

4

419.86

421.79

5

4

418.39

419.93

6

4

421.34

422.98

Table 22: Results of Ruggedness study by change in analyst.

The ruggedness of the Intermediate was carried out for change in analyst and method was found to be robust.

S. No

Parameter

SD

%RSD

1

Precision

1.331

0.317

2

Intraday precision

2.25

0.539

3

Interday precision

1.76

0.414

4

Robustness

3.122

0.395

5

Ruggedness

1.436

0.341

Table 23: Summary of Precision.

The summary of the precision is given in the above table and % RSD was found to be ≤ 2.

Accuracy

Accuracy study was performed by the recovery method. The results demonstrate that the percentage recovery in tablet due to the presence of impurity in the tablet. Percentage recovery was found to be more at higher concentration level a compare to lower concentration level (Table 24).

S. No

Drug / Formulation

Percentage Recovery

Mean

S.D.

%RSD

50%

75%

100%

1

Tablet I

99.22

101.3

103.79

101.43

2.288

2.255

2

Tablet II

99.25

101.68

103.13

101.3

1.969

1.934

Table 24: Result of recovery study by HPLC.

Limit of detection:

LOD= 3.3×Standard deviation Slope MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIZaGaaiOl aiaaiodacqGHxdaTqaaaaaaaaaWdbiaadofacaWG0bGaamyyaiaad6 gacaWGKbGaamyyaiaadkhacaWGKbGaaeiiaiaadsgacaWGLbGaamOD aiaadMgacaWGHbGaamiDaiaadMgacaWGVbGaamOBaaGcpaqaaKqzGe Gaam4uaiaadYgacaWGVbGaamiCaiaadwgaaaaaaa@5543@

LOD= 3.3×1.3313 98.44 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIZaGaaiOl aiaaiodacqGHxdaTcaaIXaGaaiOlaiaaiodacaaIZaGaaGymaiaaio daaOqaaKqzGeGaaGyoaiaaiIdacaGGUaGaaGinaiaaisdaaaaaaa@481E@

LOD=0.4462 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadseacqGH9aqpcaaIWaGaaiOlaiaaisdacaaI0aGaaGOn aiaaikdaaaa@3E5D@

Limit of quantitation:

LOQ= 10×Standard deviation Slope MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIXaGaaGim aiabgEna0cbaaaaaaaaapeGaam4uaiaadshacaWGHbGaamOBaiaads gacaWGHbGaamOCaiaadsgacaqGGaGaamizaiaadwgacaWG2bGaamyA aiaadggacaWG0bGaamyAaiaad+gacaWGUbaak8aabaqcLbsacaWGtb GaamiBaiaad+gacaWGWbGaamyzaaaaaaa@5499@

LOQ= 10×1.3313 98.44 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpjuaGdaWcaaGcbaqcLbsacaaIXaGaaGim aiabgEna0kaaigdacaGGUaGaaG4maiaaiodacaaIXaGaaG4maaGcba qcLbsacaaI5aGaaGioaiaac6cacaaI0aGaaGinaaaaaaa@4774@

LOQ=0.1352 MathType@MTEF@5@5@+= feaagKart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9 vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=x fr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbsacaWGmb Gaam4taiaadgfacqGH9aqpcaaIWaGaaiOlaiaaigdacaaIZaGaaGyn aiaaikdaaaa@3E65@

The LOD by HPLC was 446.2 ng and that of LOQ 135.2 ng the method is more sensitive and selective.

System suitability parameters

To verify that analytical system is working properly and can give accurate and precise results the system suitability parameters are to be set and it was found to be in stated range (Table 25).

Property

Values

Required Limits

Retention time (tR)

10.59

RSD ≤ 1%

Theoretical plates (N)

10224

N ≥ 2000

Resolution (R)

6.36

R ≥ 2

Table 25: System Suitability Parameters of synthesized compound.

To verify that analytical system is working properly and can give accurate and precise results the system suitability parameters are to be set and it was found to be in stated range.

Summary of method validation parameters of HPLC (Table 26).

S. No

Parameter

Observation

1

Linearity range

1-6 μg/ml

2

Slope

98.44

3

Intercept

21.54

4

Correlation coefficient

0.999

5

LOD

0.4462

6

LOQ

0.1352

Table 26: Summary of Method Validation Parameters of HPLC.

Summary of retention time and asymmetry (Table 27).

S. No

Compound

Retention Time

Asymmetry

1

Zolmitriptan

9.5

1.8

2

Impurity

10.5

1.81

3

Zolmitriptan Tablet

9.6

1.78

Table 27: Summary of retention time and asymmetry.

Quantitation of synthesized compound

Impurity was not found in bulk and in tablet I & II it was found to be 0.28%.and 0.33% respectively. As per the ICH limit the amount of impurity is more than 0.1% indicates that the impurity found in tablet formulations is potential impurity (Table 28).

S. No

Formulation

Quantisation of Impurity

1

Zolmitriptan tablet I

0.28%

2

Zolmitriptan tablet II

0.33%

Table 28: Quantisation of process related impurity of Zolmitriptan in tablets.

References

  1. Ingale SJ, Sahu CM, Paliwal RT, Vaidya S, Singhai AK (2011) Advance approaches for the impurity profiling of pharmaceutical drugs: A review. International Journal of Pharmacy and Life Sciences 2(7): 955-962.
  2. Rao NR, Nagaraju V (2003) An Overview of the recent trends in development of HPLC methods for the determination of impurities in drugs. J Pharm Biomed Anal 33(3): 335-337.
  3. Prakasha A, Nandia U, Teotiaa AK, Javed AF, Singh GN (2015) Forced Degradation Study Of Emtricitabine For Evaluation Of Genotoxic Impurity In Active Pharmaceutical Ingredient’s (API) Shelf Life. WJPPS 4(7): 1909-1919.
  4. Maggio RM, Calvo NL, Vignaduzzo SE, Kaufman TS (2014) Pharmaceutical impurities and degradation products: Uses and applications of NMR techniques. J Pharm Biomed Anal 101: 102-122.
  5. Kalyana CE, Srinivasa RVND, Sachin BS, Shailesh WG, Shailendra KS, et al. (2015) Synthesis and characterization of process related impurities of an antituberculosis drug-Prothionamide. Der Pharma Chemica 7(3): 79-84.
  6. Neelakandan K, Ashok C, Manikandan H, Santosha N, Prabhakaran B, et al. (2013) Isolation and Structural Elucidation of Novel Isomeric Process Related Impurities of Zolmitriptan. J Anal Bioanal Tech 4: 165.
  7. Sagar DS, Deshmukh VK, Chaudhari SR (2013) A Review On: Separation of different process related impurity in tablet formulation of drug. International Journal 1(7): 673-684.
  8. Shah RS, Patel MA, Naik MV, Pradhan PK, Upadhyay UM (2012) Recent Approaches of Impurity profiling in Pharmaceutical Analysis: A Review. International Journal of Pharmaceutical sciences and Research 3(10): 3603-3617.
  9. Chitturi SR, Bharath C (2008) Impurity profile study of lopinavir and validation of HPLC method for the determination of related substances in lopinavir drug substance. J Pharm Biomed Anal 48(5): 1430-1440.
  10. ICH (2008) Guidance for Industry: Q3A Impurities in New Drug Substances International Conference on Harmonisation, Genève, Switzerland, p 1-14.
  11. Bartos D, Gorog S (2008) Recent Advances in the Impurity Profiling of Drugs. Current Pharmaceutical Analysis 4(4): 215-230.
  12. Sudhakar P, Reddy GM, Reddy PP, Bhaskar BV, Mukkanti K, et al. (2006) Identification and characterization of potential impurities of Amlodipine maleate. Journal of Pharmaceutical and Biomedical Analysis 40(3): 605-613.
  13. Manish R, Kumanan R, Duganath N, Srinivasa MM, Ahmed N, et al. (2011) Synthesis, Characterization And Pharmacological Screening Of 2-Methyl-1h-Indole-3-Carboxylic Acid [2-(2- Substituted-Phenyl)-4-Oxo-Thiazolidin-3-Yl]-Amides Derivatives. International Journal of Chemical Sciences and Applications 2(1): 91-99.
© 2014-2016 MedCrave Group, All rights reserved. No part of this content may be reproduced or transmitted in any form or by any means as per the standard guidelines of fair use.
Creative Commons License Open Access by MedCrave Group is licensed under a Creative Commons Attribution 4.0 International License.
Based on a work at http://medcraveonline.com
Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version | Opera |Privacy Policy