Application of Normal and Reversed-Phase TLC/ Densitometry Methods for Determination of Naratriptan Hydrochloride in Tablet dosage form


Atul A. Shirkhedkar, Jayshri  S. Borse*, Manoj  D. Patil, Amod  S. Patil

Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, (M.S.), India 425 405

*Corresponding Author E-mail:



Naratriptan Hydrochloride (NTPH)  is used as antimigraine drugs. In the present investigations two simple, economical and precise, normal-phase  High-Performance Thin-Layer  Chromatography (Method A) and Reversed Phase  High Performance Thin Layer Chromatography (Method B) have been established for determination of NTPH  in bulk and in tablets. Method A and Method B  were developed on aluminium plates precoated with silica gel 60F254  and  RP-18 silica gel 60 F254 S  as the stationary phase, respectively.  Separation of NTPH was achieved using Chloroform: 2-propranol: triethylamine (8:1.4:0.6 v/v) in Method A and methanol: water: triethylamine (9:0.6:0.4 v/v) in Method B. Densitometry scanning were performed at 223 nm. Both methods were found to give compact spot for the NTPH at Rf 0.55 ± 0.02. Validation of Method A and Method B were performed as per as International Conference on Harmonization guidelines.






Naratriptan Hydrochloride (NTPH), N-methyl-3-(1-meth­yl-4-piperidinyl)-1H-indol-5-ethanesulfonamide  is a selec­tive 5-hydroxytryptamine 1 receptor subtype agonist and  used in the treatment of migraine and headaches [1].  NPPH is official in USP [2].


Literature survey revealed High- Performance Liquid-chromatography (HPLC) with tandem mass spectrometry [3, 4] has been reported for the determination of NTPH in biological samples. A preliminary pharmacokinetics studies on several antimigraine drugs in rabbit have been established using liquid chromatographic-electrospray-mass spectrometric [5].


A simple Spectrophotometric [6] has been established for quantification of NTPH in tablets has been reported.

High-Performance thin Layer Chromatography (HPTLC) is well known technique used for separation of drugs, phytoconstitutent. It is having wide applications over other forms of chromatographic techniques.  Several samples can be analysed at a time with less solvent consumptions [7]. From the literature survey, it has been evident that there is no TLC/densitometry method has been reported for the determination of  NTPH in bulk and in pharmaceutical formulation. Therefore, in the present endeavour an attempt has been made to estimate Naratiptan Hydrochloride in bulk and in tablet dosage form using NP-HPTLC [Method A] and RP-HPTLC [Method B] methods. Further validation of both these methods as per International Conference on Harmonization (ICH) [8].






Materials and Reagents:

Naratriptan Hydrochloride provided as a gift sample by Glaxo Smith Kline pharmaceutical Ltd. Nasik. Drugs were used without any further purification. All other reagents required for experimentation were of analytical reagent (AR) grade. For analysis, methanol, chloroform, 2- propranol and triethylamine were purchased from Merck India.


Instrumentation and chromatographic condition:

In ‘Method A’ and ‘Method B’, the samples were applied as 6 mm wide bands with the help of Linomat 5 sample applicator (Muttenz, Switzerland) fitted with a 100-μL sample syringe (Hamilton, Bonaduz, Switzerland). Chromatography was performed on 20 cm ×10 cm aluminium-backed HPTLC plates precoated with 200-μm layers of silica gel 60 F254S in ‘Method A’ while it was performed  on 20 cm × 10 cm aluminium-backed HPTLC plates precoated with 200-μm layers of silica gel 60 RP-18 F254S in ‘Method B’. The plates were previously washed with methanol before chromatographic measurements and activated at 1050C for 5 min. The plates were developed in a pre-saturated Camag twin trough glass chamber (20 cm × 10 cm). In Method A and Method B, chloroform: 2-propanol: triethylamine (8:1.4:0.6 v/v) and methanol: water: triethylamine (9:0.6:0.4 v/v), respectively were used as mobile phases with optimized chamber saturation time 15 min and 30 min, respectively. The plates were developed to a distance of 8.0 cm and scanned densitometrically using Camag TLC Scanner 3 equipped with winCATS software version 1.3.0 at 223 nm for both methods. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200- 400 nm. Evaluation was performed using peak area with linear regression.


Preparation of solutions standard and calibration curve:

Stock standard solution was prepared by dissolving 25 mg of NTPH in 25 ml of methanol to obtain concentration 1 mg/mL.  From this stock solution, 1- 6 mL were transferred into six different 10 ml volumetric flask and volume was made up to the mark. An appropriate volume 10 µL of Naratriptan Hydrochloride was applied on HPTLC and RP-HPTLC plates separately with the help of microlitre    syringe, using Linomat 5 sample applicator to obtain the concentration of 1000, 2000, 3000, 4000, 5000 and 6000 ng/band, respectively. The calibration curves were studied also for  within day and day-to-day reproducibility. Each experiment was repeated six times.


Application of the proposed method for estimation of Naratriptan Hydrochloride in tablets:

Twenty tablets (Naratrex; Label claim 1 mg) were weighed; average weight was determined and then crushed in to fine powder. An accurately weighed tablet powder equivalent one tablet was  transferred to 10 mL volumetric flask containing 10 mL methanol, sonicated for 10 min, volume was adjusted to mark and filtered using 0.45 µm filter (Mill filter, Milford, MA). A volume of 20 µL, were applied on HPTLC and RP-HPTLC plates for assay of NTPH. The plates were developed and scanned as described in above chromatographic conditions in both ‘Method A’ and ‘Method B’.



Optimization of HPTLC method:

a)      Method A:

Several solvent mixtures in different ratios were tested to obtain a compact band of Naratriptan Hydrochloride. chloroform: 2-propanol: triethylamine (8:1.4:0.6 v/v) was found to give a compact band for Naratriptan Hydrochloride with an 𝑅𝑓 value 0.55 ± 0.02. This mobile phase gave good resolution for the separation of Naratriptan Hydrochloride. Chamber saturation with mobile phase for 15 min was found to be resolution. The chromatogram shown in Figure 1.



Figure 1: Densitogram of Naratriptan Hydrochloride standard (Rf 0.55 ± 0.02)


measured at 223 nm.

b)      Method B:

The spot was developed in mixtures of methanol and water in the ratio of 9.4:0.6 v/v. The Rf value obtained was good but slight tailing was observed. Hence, to reduce the tailing, triethylamine was added in the solvent system. Thus, the final mobile phase consisted of methanol: water: triethylamine in the ratio (9:4: 0.6: 0.4 v/v). The chamber saturation time was 30 min. The Rf for Naratriptan were found to be 0.55 ± 0.02 (Figure 2).


Figure 2: Densitogram of Naratriptan Hydrochloride standard (Rf 0.55 ± 0.02) measured at 223 nm


Validation of method:

The method was validated by establishing linearity, accuracy, inter - day and intra - day precision of measurement of sample application. The limit of detection and limit of quantification were also determined.



Linearity was studied in the concentration range from 1000 – 6000 ng per band for both methods. The drugs showed good linearity in the tested range. The regression co-efficient values for Naratriptan Hydrochloride were found to be r2 > 0.99 in both ‘Method A’ and ‘Method B’.


The accuracy of the experiment was established by spiking pre-analyzed sample with known amounts of the corresponding drugs at three different concentration levels i.e. 80, 100 and 120 % of the drug in the tablet. The spiked samples were then re-analyzed for three times. The mean recovery is within acceptable limits, indicating both methods are accurate (Table 1).



Precision of the method was studied as repeatability and intra-day and inter-day variations. The repeatability of sample application and measurement of peak area was determined by performing six replicate measurements of 3000 ng/band for NTPH; the effects on the results were studied in terms of %RSD and found to be less than 2.

Intra-day variation was determined by analyzing three different concentrations for three times within a day and Inter-day precision was assessed by three different concentrations for three different days, over a period of week.


The intra-day and inter-day variation were measured at three different concentrations 2000, 3000, 4000 ng/band of NTPH. The effects on results of intra-day and inter-day variations were assessed in terms of %RSD; found to be less than 2.





Table 1: Recovery studies



Initial amount (ng / band)

Amount added (%)

  % recovery

%RSD [n=3]


Method A

Naratriptan Hydrochloride














Method B


Naratriptan Hydrochloride















a)      Method A:

The specificity of the method was ascertained by analyzing drug standards and sample. The mobile phase resolved the drug very efficiently. The Rf value of Naratriptan Hydrochloride was found to be 0.55. The peak purity of Naratriptan Hydrochloride extracted from tablet and standard Naratriptan Hydrochloride was tested at the peak - start (S), peak - apex (A) and at the peak - end (E) position (Figure 3).



Figure 3: Peak purity spectra of Naratriptan Hydrochloride standard 1, sample 2 extracted from Naratriptan Hydrochloride tablet, scanned at the peak-start, peak-apex and peak-end positions of the band (Correlation > 0.99).

b) Method B:

The mobile phase designed for the method resolved both the drugs very efficiently. The Rf value of Naratriptan Hydrochloride was found to be 0.55. The peak purity of Naratriptan Hydrochloride extracted from tablet and Naratriptan Hydrochloride standard was tested at the peak - start (S), peak - apex (A) and at the peak - end (E) positions (Figure 4).



Figure 4: Peak purity spectra of Nara standard 1, sample 2 extracted from Naratriptan Hydrochloride tablet, scanned at the peak - start, peak - apex and peak - end positions of the band (Correlation > 0.99).


Ruggedness and Robustness:

Ruggedness of the both method was performed for Naratriptan Hydrochloride by two different analysts maintaining similar experimental and environmental conditions.       Robustness of the method was performed by introducing various changes in the previous chromatographic conditions; effects on the results were examined for both method.



The sensitivity of measurements of Naratriptan by the use of the proposed method was estimated in terms of the Limit of Quantitation (LOQ) and the lowest concentration detected under the chromatographic conditions as the Limit of Detection (LOD).


LOQ and LOD were calculated by the use equation LOD = 3.3 x N/B and LOQ = 10 x N/B,


where ‘N’ is standard deviation of the peak areas of the drugs (n=3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve. The results were recorded for both the methods.


Different validation parameters for the both methods for determining Naratriptan Hydrochloride content were summarized in Table 2.





Table 2: Summary of Validation parameters


Method A

Method B


(correlation coefficient)









Ruggedness [% RSD]



Analyst -I [n = 6]



Analyst -II [n = 6]



Robustness [% RSD] [n = 6]



Mobile phase composition



Duration of saturation time



Mobile phase volume



Development distance






Limit of Detection [ng]



Limit of Quantitation [ng]



Precision [%RSD]



Intra-day [n = 3]

0.13- 0.77

0.34 - 0.71

Inter-day [n = 3]

0.17- 0.63

0.48 - 1.20

Repeatability [n = 6]






Analysis of tablet formulation:

In ‘Method A’ and ‘Method B’, compact spots of Naratriptan Hydrochloride obtained with Rf values 0.55. In both these methods good separation and well resolved spots were obtained which indicate that there is no interferences commonly present in the tablet formulation shown in Table 3.


Table 3: Analysis of Tablet formulation



Label Claim (mg)

%Amount Found


 (n = 5)


Naratriptan Hydrochloride








Naratriptan Hydrochloride







n- no of estimation



The proposed ‘Method A’ and ‘Method B’ provide simple, accurate and reproducible quantitative analysis for determination of Naratriptan Hydrochloride in bulk and tablet dosage form. Both these method were validated according to ICH guidelines.



The authors are thankful to R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur Dist: Dhule (MS) 425 405



1.       Budavari S. The Merck Index. 14th ed. , USA: Merck and Co., Inc.; NJ 2001. p. 6420

2.       United States Pharmacopoeia (USP 29 NF 24), United States Pharmacopoeial Convention, Inc., Rockville, p. 1340, 2005.

3.       Reddy BC., Bahlul ZSA., BabuRao C., Shaik RP. Method development and validation for naratriptan Hydrochloride determination in human plasma by HPLC with tandem mass spectrometry detection, and its application to bioequivalence study. Brazil J. Pharma. Sci. vol. 2011; 47(1):

4.       Vishwanathan, K., Bartlett MG., Stewart JT. Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom., v.14, n.3, p.168-172, 2000.

5.       Dulery BD., Petty MA., Schoun J., David M., Huebert ND., A method using a liquid chromatographic-electrospray-mass spectrometric assay for the determination of antimigraine compounds: preliminary pharmacokinetics of MDL 74,721, sumatriptan and naratriptan, in rabbit. J. Pharm. Biomed. Anal., 1997:15(7), 1009-1020,.

6.       Kumara Swamy. G, JMR.Kumar, J.V.L.N Sheshagiri Rao, U.Ashok Kumar, Vidya sagar. P. Spectrophotometric Determination of Naratriptan Hydrochloride in Bulk and Pharmaceutical Dosage Form. Indo. Ame. J. Pharm Res. 2011; 1(4):253-256.

7.       Attimarad, M., Ahmed, K. M., Aldhubaib, B. E., & Harsha, S. (2011). High-performance thin layer chromatography: A powerful analytical technique in pharmaceutical drug discovery. Pharmaceutical methods2(2), 71-75.

8.       ICH-Guidelines Q2 (R1), Validation of Analytical Procedures: Text and Methodology. (2005).




Received on 10.04.2018       Accepted on 21.05.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(2):91-95.

DOI: 10.5958/2231-5675.2018.00018.2