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Rapid Simultaneous Determination of Sumatriptan Succinate and Naproxen Sodium in Combined Tablets by Validated Ultra Performance Liquid Chromatographic Method
ISSN: 2155-9872
Journal of Analytical & Bioanalytical Techniques

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Rapid Simultaneous Determination of Sumatriptan Succinate and Naproxen Sodium in Combined Tablets by Validated Ultra Performance Liquid Chromatographic Method

Yarram Ramakoti Reddy1*, Kakumani Kishore Kumar1, MRP Reddy2 and K. Mukkanti1

1Centre for Chemical Science and Technology, Institute of science and Technology, Jawaharlal Nehru Technological University, Hyderabad 500085, India

2Centre for Meterials for Electronics Technology IDA, HCL post, Cherlapally, Hyderabad, India

*Corresponding Author:
Dr. Yarram Ramakoti Reddy
Centre for Chemical Science and Technology
Institute of science and Technology
Jawaharlal Nehru Technological University, Hyderabad 500085, India
Tel: (+)91 9849392039
E-mail: [email protected], [email protected]

Received date: April 18, 2011; Accepted date: May 28, 2011; Published date: May 30, 2011

Citation: Reddy YR, Kumar KK, Reddy MRP, Mukkanti K (2011) Rapid Simultaneous Determination of Sumatriptan Succinate and Naproxen Sodium in Combined Tablets by Validated Ultra Performance Liquid Chromatographic Method. J Anal Bioanal Tech 2:121. doi: 10.4172/2155-9872.1000121

Copyright: © 2011 Reddy YR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

A stability- indicating ultra perfomance liquid chromatography (UPLC) method was developed for the simultaneous determination of sumatriptan succinate and Naproxen sodium in pharmaceutical dosage forms. The chromatographic separation was achieved on C18, 50 mm × 4.8 mm, 1.8-μm particle size column. The mobile phase contains a mixture of 0.2% ortho phosphoric acid and acetonitrile as the mobile phase in gradient elution technique. The retention time of Sumatriptan and Naproxen was found to be 1.7 and 2.7 min respectively. The total runtime was 5 min within which two active compounds and degradation products were separated. This method allows the determination of 850-2565 μg mL -1 of sumatriptan succinate and 5000-15000 μg mL -1 of Naproxen sodium. The flow rate was 1.0 mL min -1 and the detection wavelength was 225 nm. The limit of detection (LOD) for sumatriptan succinate and Naproxen sodium was 1.9 and 1.5 μg mL -1 , respectively. The limit of quantification (LOQ) for sumatriptan succinate and Naproxen sodium was 6.3 and 4.8 μg mL -1 , respectively. This method was validated for accuracy, precision, linearity and robustness. sumatriptan succinate and Naproxen sodium were subjected to different ICH prescribed stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation and the method was also found to be stability indicating.

Keywords

Method development and validation; Simultaneous sumatriptan succinate; Naproxen sodium stability-indicating.

Introduction

Sumatriptan succinate (SS) is a selective 5-hydroxytryptamine (5-HT) receptor subtype agonist. Chemically it is known as 3-[2-(dimethylamino) ethyl]-N-methyl-indole-5-methanesulfonamide succinate (1:1). The empirical formula is C14H21N3O2S•C4H6O4, representing a molecular weight of 413.5. It is indicated for indicated for the acute treatment of migraine attacks with or without aura in adults.

Naproxen sodium (NS) is a member of arylacetic acid group of nonsteroidal anti-inflammatory drugs (NSAIDS). Chemically it is (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, sodium salt. The empirical formula is C14H13NaO3, representing a molecular weight of 252.23.

A tablet formulation containing 85 mg of Sumatriptan succinate and 500 mg of Naproxen sodium has recently been approved for the acute treatment of migraine. The combination product was proved to have superior efficacy compared to its individual components for the acute treatment of migraine. Sumatriptan, work early in the migraine process at the trigeminovascular unit as agonists of the serotonin receptors (5-HT receptors) 1B and 1D. They block vasoconstriction and block transmission of signals to the trigeminal nucleus and thus prevent peripheral sensitization. The analgesic effect of Naproxen sodium helps relieve the headache, while the anti-inflammatory effect decreases the neurogenic inflammation in the trigeminal ganglion, thus preventing the development of central sensitization.

So far, several liquid chromatography procedures have been described for the determination of SS and NS [1-19]. But, these procedures were developed to estimate either SS or NS individually and in combination with other drugs from formulation, plasma, urine, intestinal perfusion samples and in bulk drugs. For simultaneous determination of SS and NS in formulation, there are two spectrometric methods [20-21] and an HPTLC [22] method was reported. Whereas no single liquid chromatographic (LC) method has been reported for their simultaneous estimation from the combined tablets. Hence, it is necessary to develop a rapid, accurate and validated LC method for the simultaneous determination of SS and NS from combined dosage form.

Ultra performance liquid chromatography (UPLC) is a recent technique in liquid chromatography, which enables significant reductions in separation time and solvent consumption. Literature indicates that UPLC system allows about ninefold decrease in analysis time as compared to the conventional HPLC system using 5μm particle size analytical columns, and about threefold decrease in analysis time in comparison with 3μm particle size analytical columns without compromise on overall separation [23-27].

Hence, a rapid, accurate stability indicating UPLC method for the simultaneous determination of SS and NS from combined dosage form was developed and validated.

Experimental

Instrumentation and chromatographic conditions

The Waters UPLC Acquity system we used consists of a binary solvent manager, a sample manager and a UV detector. Zorbax SB C-18, 50 mm x 4.6 mm i.d with 1.8μm particles was used as stationary phase. 0.2% ortho phosphoric acid in water (pH 2.2) as solvent A and acetonitrile and water in the ratio 90:10; v/v, was as solvent B used for mobile phase. Prior to use, the mobile phase was mixed thoroughly and degassed. The gradient program was set as B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5. The mobile phase pumped at 1.0 mL min-1. The eluants were monitored at 225nm. The injection volume for samples and standards were 2μL. Methanol, acetonitrile and water in the ratio, 40:40:20; v/v/v, respectively was used as diluent.

Reagents

Standards were supplied by D.C.O. Hyderabad, India. HPLC grade acetonitrile, analytical grade ortho phosphoric acids were purchased from Merck (Mumbai, India). Water was prepared by Millipore MilliQ Plus water purification system. Commercial pharmaceutical preparation of Triximet combined tablets were purchased from the market. The declared content of tablets was Sumatriptan 85 mg and Naproxen 500 mg per tablet.

Preparation of solutions

Standard solutions: A standard solution containing 68μg/mL-1 of SS and 400μg/mL-1 of NS were prepared by dissolving appropriate amount of SS and NS in diluent. All the solutions were covered with aluminium foil to prevent photolytic reaction until the time of analysis.

Sample preparation: Ten tablets, each containing 85 mg of SS and 500 mg of NS were dissolved in 500 mL diluents to get 1710μg mL-1 of SS and 10000μg mL-1 of NS. 4 mL of above solution was diluted to 100 mL to get 68μg mL-1 of SS and 400μg mL-1 of NS. The solution was filtered through 0.45 μm Millipore PVDF filter. Then 2μL of these solutions were injected in the column and chromatogram was. The retention times of SS and NS were found to be 1.8 min and 2.6 min, respectively.

System suitability solution criteria

The system suitability was assessed by five replicate analyses of the drugs at concentrations of 68μg mL-1 of SS and 400μg mL-1 of NS. The acceptance criteria was not more than 2.0% for the RSD for the peak areas and not more than 2.0 for tailing factor for the peaks of the both the drugs.

Method validation

Method validation was performed as per ICH guidance [28-29] for simultaneous determination of SS and NS in the formulations. The following validation characteristics were addressed: linearity, detection limit, quantification limit, precision, accuracy and specificity.

System suitability criteria

The system suitability test solution was injected and the chromatographic parameters like relative standard deviation for replicate injections of both SS and NS and the tailing factor for SS and NS peaks were evaluated for proving the system suitability.

Specificity–forced degradation studies

Forced degradation studies were performed on SS and NS combined tablets to prove the stability indicating property of the method. The stress conditions employed for degradation study of SS and NS include light exposure [29], heat (105°C), acid hydrolysis (1 N HCl at 50°C), base hydrolysis (1N NaOH at 50°C), water hydrolysis at 50°C and oxidation (1% H2O2 at 30°C). For light studies, the monitoring period was 10 days whereas for heat, acid, base and water hydrolysis it was 48 h. Oxidation was carried out for 24h. Peak purity of the principal peak in the chromatogram of stressed samples of SS and NS tablets was checked using photo diode array detector.

Linearity of response

Linearity solutions were prepared from stock solution at five concentration levels from 34μg mL-1 to 102μg mL-1 for SS and from 200μg mL-1 to 600μg mL-1 for NS. The slope, Y-intercept and correlation coefficient were calculated.

Precision

Repeatability (intra-day): The precision of the assay method was evaluated by carrying out six independent assays of SS and NS (1.71 mg mL-1 of SS and 10.0 mg mL-1 of NS) test samples against qualified reference standard. The percentage of RSD of six assay values was calculated.

Intermediate precision (inter-day): Different analyst from the same laboratory evaluated the intermediate precision of the method. This was performed by assaying the six samples of SS and NS tablets against qualified reference standard. The percentage of RSD of six assay values was calculated.

Accuracy (Recovery study)

Recovery of the assay method for SS and NS was established by three determinations of test sample using tablets at 50%, 100% and 150% of analyte concentration (1.71 mg mL-1 of SS and 10.0 mg mL-1 of NS). Each solution was injected trice (n=3) into HPLC system and the average peak area of S and NS peaks was calculated.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ for SS and NS were estimated at a signal-to noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentration.

Robustness

To determine the robustness of the method the experimental conditions were deliberately changed and the resolution of SS and NS, tailing factor and % RSD for five replicate injections was evaluated. The mobile phase flow rate was 1.0 mL min−1; to study the effect of flow rate on resolution it was changed to 0.9 and 1.1 mL min−1. The effect of pH was studied at pH 2.1 and 2.3 (instead of pH 2.2). The effect of column temperature was studied at 25 and 35°C (instead of 30°C). In all these experiments the mobile phase components were not changed.

Solution stability and mobile phase stability

The stability of SS and NS in solution was determined by leaving test solutions of the sample and reference standard in tightly capped volumetric flasks at room temperature for 48h during which they were assayed at 24h intervals. Stability in the mobile phase was determined by analysis of freshly prepared sample solutions at 24h intervals for 48h and comparing the results with those obtained from freshly prepared reference standard solutions. The mobile phase was prepared at the beginning of the study period and not changed during the experiment. The RSD (%) of the results was calculated for both the mobile phase and solution-stability experiments.

Method development and optimization of stability indicating assay method

The method was optimized to separate major degradation products formed under varies stress conditions from SS and NS. The main target of the chromatographic method is to get the separation for closely eluting degradation products, mainly for the degradation product at 3.18 RT, which is eluting very closely to the NS. The degradation samples were run using different stationary phases like C18, C8 and Mobile phases containing buffers like phosphate and acetate with different pH (2-7) and using organic modifiers like acetonitrile and methanol in the mobile phase. But the separation was satisfactory in the adopted chromatographic conditions only. It indicated that the gradient elution with 0.2% ortho phosphoric acid in water as solvent A and acetonitrile and water in the ratio 90:10; v/v, was as solvent B for mobile phase was successful in separating drugs and all chromatographic degradation products (Figure 2). The detailed experimentation is reported in Table 1.

Trial no. UPLC
Conditions
  Remark
1 Column Inertsil ODS-3, 50 mm × 2.1 mm, 2 μm particles Separation of unknown impurity (RT 3.18)  from NS is poor
Mobile phase Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2) Solvent B: Acetonitrile and water in the ratio 90:10 v/v
Flow rate 1.0  mL min-1ww
Gradient Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5
2 Column Waters AQUITY BEH C8, 50 mm × 2.1 mm, 1.7 μm particles Separation of unknown impurity (RT 3.18)  from NS is poor
Mobile phase Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2)
Solvent B: Acetonitrile and water in the ratio 90:10 v/v
Flow rate 1.0  mL min-1
Gradient Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5
3 Column Mobile phase   Flow rate
Gradient
Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles NS Peak is strongly retained and tailing of NS is more (1.8)
Solvent A: 0.01 M KH2PO4 buffer, tetra hydro furan and methanol in the ratio 67: 23: 10 (v/v/v), pH adjusted to 4.0 with ortho phosphoric acid.
Solvent B: Acetonitrile and water in the ratio 90:10 v/v
0.6 mL min-1
Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5 NS Peak is strongly retained and tailing of NS is more (1.9)
4 Column Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles
Mobile phase Solvent A: 0.01 M ammonium acetate buffer pH adjusted to 7.0 with aqueous ammonia solution
Solvent B: Acetonitrile and water in the ratio 90:10 v/v
Flow rate 1.0  mL min-1
Gradient program Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5
5 Column Zorbax SB C-18 column, 50 mm × 2.1 mm, 1.8 μm particles Separation of unknown impurity (RT 3.18)  from NS is satisfactory and tailing for both SS and NS peaks were less than 1.3
Mobile phase Solvent A: 0.2% ortho phosphoric acid in water (pH 2.2)
Solvent B: Acetonitrile and water in the ratio 90:10 v/v
Flow rate 1.0  mL min-1
Gradient program Time (min) / % solution B: 0/5, 2/95, 4/95, 4.1/5, 5.0/5

Table 1: Results from different method development trials.

Results and Discussion

Method validation

Validation of an analytical procedure is the process by which it is established, by laboratories studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications [28].

System suitability

The system suitability test solution was injected and the chromatographic parameters like relative standard deviation for replicate injections of I and DC and the tailing factor for both SS and NS peaks are evaluated. The relative standard deviation for replicate injections of both SS and NS was 0.5% and 0.3% respectively. The tailing factor for both SS and NS peaks was 1.2% and 1.3%, respectively. This indicates the suitability of the system.

Linearity of response

Calibration curve obtained by least square regression analysis between average peak area and the concentration showed (Table 2a and Table 2b) linear relationship with a regression coefficient of 0.999. The best fit linear equation obtained was Y =8620 Con - 5872 for SS and Y=11070 Con - 13451 for NS. Analysis of residuals indicated that the residuals were normally distributed around the mean with uniform variance across all concentrations suggesting the homoscedastic nature of data. Selected linear model with univariant regression showed minimum % bias indicating goodness of fit which was further supported by the low standard error of estimate and mean sum of residual squares.

Concentration in
µg/ml
Mean area Response achieved Response calculated thru Trend line equation Residual (Response practical -Response theoretical) Residual square
34.2 294426 288932.80 -5493.2 98899047.0
51.3 430896 436335.20 5439.2 1133160.3
68.4 580852 583737.60 2885.6 207613517.4
85.5 731256 731140.00 -116.0 200366856.0
102.6 881258 878542.40 -2715.6 308163981.2
Residual sum of squares 75474769.6
Correlation coefficient 0.999
Trend line equation        y = 8620x - 5872

Table 2a: Residual summary of Linearity results of Sumatriptan succinate

Concentration in µg/ml Mean area Response achieved Response calculated thru Trend line equation Residual (Response practical -Response theoretical) Residual square
203.2 2245986 2236041.20 -9944.8 98899047.0
304.8 3361852 3360787.50 -1064.5 1133160.3
406.4 4471125 4485533.80 14408.8 207613517.4
508 5596125 5610280.10 14155.1 200366856.0
609.6 6752581 6735026.40 -17554.6 308163981.2
Residual sum of squares 816176561.9
Correlation coefficient 0.999
Trend line equation y = 11070x - 13451

Table 2b: Residual summary of Linearity results of Naproxen sodium

Precision

The precision of an analytical method gives information on the random error. It expresses of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. The percentage RSD values for the precision study was 0.7%, 0.4% (inter-day precision) and 0.6%, 0.7% (intra-day precision) for SS and NS, respectively. This is confirming good precision of the method (Table 3).

S.No Parameter Variation % RSD
for Assay
Sumatriptan succinate Naproxen sodium
1 Repeatability(inter-day) (a)  Analyst-1(b) Waters Acquity UPLC system with PDA detector.
(c)  Day-1
0.7 0.4
2 Intermediate precision
(intra-day)
(a)  Analyst-2(b) Waters Acquity UPLC system with tunable UV detector.
(c)  Day-2
0.6 0.4

Table 3: Precision results

Accuracy-recovery test

The percentage recovery of SS was ranged from 98.2 to 100.2 and NS was ranged from 99.8 to 101.6. Excellent recoveries were made at each added concentration (Table 4).

S.No Concentration (%) Mean recovery (%)  (n = 3) % RSD
SS NS SS NS
1 50 99.8 99.9 0.31 0.22
2 100 99.3 101.0 0.20 0.28
3 150 100.3 100.2 0.25 0.26

Table 4: Recovery study

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection of SS and NS was 1.9 and 1.5μg /mL-1, respectively for 2μL injection volume. The limit of quantification of SS and NS was 6.3 and 4.8μg /mL-1, respectively for 2μL injection volume.

Robustness

When mobile phase flow rate, pH and column temperature were deliberately varied resolution between SS and NS was greater than 3.0, tailing factor and % RSD for five replicate injections of SS and NS was less than 1.5, illustrating the robustness of the method (Table 5).

Parameter Temperature
(± 5°C of set temperature)
Flow rate
(± 0.1 ml/min of the set flow)
pH
(± 1 unit)
Variation 20°C 30°C 0.6 ml/min 0.8 ml/min 95% 105%
% RSD for 5
replicate injections
SS 1.3 1.2 1.4 1.1 1.2 1.2
NS 1.2 1.2 1.2 1.1 1.3 1.2
USP Resolution between SS & NS 5.2 5.8 5.5 5.7 5.0 5.9

Table 5: Robustness Study

Stability in solution and in the mobile phase

RSD (%) for assay of SS and NS during solution stability and mobile phase stability experiments was within 0.9%. No significant changes in the amounts of the two drugs were observed during solution stability and mobile phase experiments. The results from solution stability and mobile phase stability experiments confirmed that standard solutions and e mobile phase were stable for up to 48 h during assay determination (Table 6).

S.No Interval % Assay
Solution stability
% Assay
Mobile phase stability
1 0 h 99.4 98.2
3 24 h 99.0 98.1
5 48 h 98.2 98.3
% RSD 0.6 0.1

Table 6: Solution and mobile phase stability results

Specificity–forced degradation studies

Degradation was not observed in SS and NS stressed samples that were subjected to light, heat, water and oxidation. However, the degradation was observed under base hydrolysis and acid hydrolysis. The peak purity test results derived from PDA (Photo Diode Array detector) confirmed that the SS and NS peaks were pure and homogeneous in all the analyzed stress conditions. This indicates that the method is specific and stability indicating (Figure 2 and Table 7).

Stress condition Time ~% Degradation
Acid hydrolysis
(1N HCl,50°C )
24 h 11.2 %
Base hydrolysis
(1N NaOH, 50°C )
24 h 5.3 %
Oxidation (1% H202) 24 h 0.4%
Water hydrolysis (50°C) 24 h 0.5%
Thermal (105°C) 24 h 0.3%
Light  (photolytic
degradation)
10 days 0.2%

Table 7: Forced degradation results.

analytical-bioanalytical-techniques-Naproxen-sodium

Figure 1: Chemical structures of Naproxen sodium and Sumatriptan succinate.

analytical-bioanalytical-techniques-Sumatriptan-succinate

Figure 2: A typical chromatograms obtained from Naproxen sodium and Sumatriptan succinate tablets and from stressed samples.

Conclusion

A simple specific stability indicating liquid chromatographic method is developed for the quantification of SS and NS simultaneously in combined dosage forms. This method is validated and it is found to be specific, precise, accurate, robust and linear for the detection and quantification of SS and NS. The method is stability-indicating and can be used for routine analysis of production samples and to check the stability of samples of SS and NS simultaneously in combined dosage forms.

Acknowledgement

The authors are very grateful to C-MET for their continuous support for this research work.

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