Method development and validation of RP-HPLC method for the determination of sumatriptan in bulk and pharmaceutical dosage form

 

Rakam Gopi Krishna¹*, M. Srinivasa Murthy¹, V. Kavya²

¹Department of Pharmaceutical Analysis, Vignan Institute of Pharmaceutical Sciences, Deshmukhi, Pochampally, Hyderabad. Telangana 508284 India.

²Department of Pharmaceutical Analysis, Chaitanya College of Pharmacy Education & Research, Hanamkonda, Warangal, Telangana 506001 India.

 

ABSTRACT:

The objective of the study was to develop RP-HPLC method for the determination of purity of sumatriptan in bulk and pharmaceutical dosage form. The development of an analytical method for the determination of drugs by HPLC has received considerable attention in recent years because of their importance in quality control of drugs and drug products. The aim of this current study was to develop a simple, rapid, precise, accurate and sensitive HPLC method for the analysis of sumatriptan in bulk and its pharmaceutical dosage form by using solvent system of TEA : ACN : methanol in the ratio 80:10:10 and C₈  ODS Inertsil (250*4.6mm, 5mi.d) stationary phase. The chromatographic condition is set at flow rate of 1ml/min with PDA detector at 221 nm. As per ICH requirements validation studies are carried out by using freshly prepared solutions. The linearity was demonstrated over the concentration range of 5-150 µg / ml and value was found to be as 0.99998. The %RSD of precision was found to be 0.260. The LOD and LOQ were found to be 1.967μg/ml and 5.961μg/ml respectively. Forced degradation studies were carried out under various stress conditions to demonstrate the stability-indicating capability of the developed RP-HPLC method. The proposed method was found to be simple, precise, accurate and validated according to the International Council for Harmonisation guidelines.

 

 

INTRODUCTION:

Pharmaceutical analysis is defined as the branch of practical chemistry which deals with the resolution, separation, identification, determination and purification of a given sample of a pharmaceutical dosage form, the detection and estimation of impurities that may be present there in is also included¹. Analytical chemistry is the science to analyze morphologies, compositions and quantities of analytical targets. These analytical results have played critical roles from the understanding of basic science to a variety of practical applications, such as biomedical applications, environmental monitoring, quality control of industrial manufacturing and forensic science².

 

Validation is the process of establishing documentary evidence demonstrating that a procedure, process or activity carried out in testing and then production maintains the desired level of compliance at all stages.³ The validation of the method was based on FDA guidelines and on standard bioanalytical method validation recommendation.⁴ The goal of equipment validation is to produce constant result with minimal variation without compromising the product and performance of the equipment.

 

Instrumental method is an exciting and fascinating part of chemical analysis that interacts with all areas of chemistry and with many other areas of pure and applied sciences. Analytical instrumentation plays an important role in the production and evaluation of new products and in the protection of consumers and environment.⁵ This instrumentation provides lower detection limits required to assure safe foods, drugs, water and air. Instrumental methods are widely used by Analytical chemists to save time, to avoid chemical separation and to obtain increased accuracy.⁶

Sumatriptan is chemically 1-{3-[2-(dimethylamino) ethyl]-1H-indol-5-yl}N methyl methane sulphonamide. It is a vasoconstrictor agent & selective serotonin agonist, freely soluble in water. Adenylate cyclase, activity is inhibited via regulatory G proteins, increases intracellular calcium, and affects other intracellular events. It should not be given to patients who have had myocardial infarction or have ischaemic heart disease. The Mechanism of action of drug is the 5-HT_1B and 5-HT_1D receptors function as autoreceptors, which inhibit the firing of serotonin neurons and a reduction in the synthesis and release of serotonin upon activation. After sumatriptan binds to these receptors, adenylatecyclase activity is inhibited via regulatory G proteins, increases intracellular calcium, and affects other intracellular events. This results in vasoconstriction and inhibtion of sensory nociceptive (trigeminal) nerve firing and vasoactive neuropeptide release.

 

 

Structure of Sumatriptan:

Limited UV methods were described for sumatriptan in combination⁷, visible⁸ spectrophotometric methods were also reported for sumatriptan succinate in bulk and pharmaceutical dosage forms. The drug directly reaches to the systemic circulation through the internal jugular vein and bypasses the drugs from the hepatic first pass metabolism, which leads to high bioavailability.¹¹

 

Several analytical techniques like HPLC^12-18, HPLC-MS-MS^19-22, HPLC- ECD^23-24, HPLC-coulometry²⁵, capillary LC-MS-MS²⁶, HPTLC²⁷, spectrophotometric with HPTLC²⁸, RP-HPLC with colorimetric²⁹, UV³⁰, voltametry³¹, capillary electrophoresis³², and densitometry with spectrophotometric detection³³ have been reported in the literature.

 

The developed RP-HPLC method was validated with respect to linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantification. The method was validated as per the guidelines of the International Council for Harmonisation guidelines (ICH).³⁴

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Sample of sumatriptan was supplied by Spectrum pharma research solutions (Hyderabad). Methanol, acetonitrile and potassium dihydrogen phosphate were obtained from Rankem. High purity water was attained by using Millipore Milli Q Plus water purification system.

Chromatographic conditions:

The method was developed by using Phenomenex kinetex (250*4.6mm, 5mi.d) column with a mobile phase comprising a mixture of Acetonitrile: Methanol: Tri ethyl amine (PH-2.5 with OPA)-10:10:80. The flow rate was maintained at 1 ml/min. The column temperature was maintained at 30°C and the eluted compounds were monitored at the wavelength of 221 nm. The sample injection volume was 20ml.

 

Preparation of diluents:

The diluent used for the analysis was selected based on the solubility of the drug. Mobile Phase was used as diluent.

 

Preparation of Buffer:

Tri ethyl amine 1ml was dissolved into 1 litre water and adjusted the pH to 2.5 with ortho Phosphoric acid.

 

Mobile phase:

A mixture of buffer, acetonitrile and methanol was prepared in the ratio of (80:10:10). It was filtered and degassed.

 

Preparation of standard stock solution:   

About 25 mg of sumatriptan working standard was weighed accurately and transferred into a 100 ml volumetric flask. 70 ml of diluent was added, sonicated to dissolve and diluted to volume with diluent. Further 5 ml of the solution was taken and diluted to 50 ml with the diluent.

 

Preparation of sample solution:

Tablets 5 were accurately weighed and the average weight of each tablet was calculated and tablets were crushed. The powder equivalent to one tablet was taken into a 100 ml volumetric flask. 70 ml of diluents was added, sonicated to dissolve and diluted to volume with diluent. Further 5 ml of the solution was taken and diluted to 50 ml with the diluent. Later, it was filtered through 0.45μ nylon syringe filter.

 

Method development:

Many trials have been performed by various mobile phases, flow rate and stationary phase. After observing the theoretical plates and stability factor, various chromatographic parameters were chosen. The mobile phase composition acetonitrile: methanol: tri ethyl amine (pH-2.5 with OPA)-10:10:80 with Phenomenx kinetex column was used with detection of wavelength of 221 nm with isocratic elution with run time of 10 min with ambient temperature with Rt 5.13.

 

Method validation:

The method was validated for linearity, precision, accuracy, robustness and ruggedness, according to ICH guidelines.⁹

 

System suitability:

The stock solution of sumatriptan standard was injected six times into HPLC system as per test procedure. The system suitability parameters were evaluated from standard chromatograms were obtained, the % RSD of retention times; tailing factor, theoretical plates and peak areas were calculated from six replicate injections. System suitability parameters should be met.

 

Linearity:

The linearity of the method was demonstrated over the concentration range of 5-150 µg / ml of the target concentration. From the stock solution aliquots of 0.25 ml, 0.5 ml, 1.25ml, 2.5ml, 3.75ml, 5ml, 6.25ml, 7.5ml  in a 50 ml volumetric flask and diluted up to the mark with mobile phase to get the concentration of 5, 10, 25, 50, 75, 100, 125, 150 ppm respectively. The solutions were injected in to HPLC system as per test procedure. A calibration curve was plotted for concentration v/s peak area.

 

Acceptance criteria:

1.     Correlation coefficient should not be less than 0.9990.

2.     % RSD of peak areas for solutions 1, 2, 3, 4, 5, 6, 7 should not be more than 2.0 %.

 

Precision:

A)   System precision (Repeatability):

From the sample stock solution a sample solution of 25 ppm is prepared and it was injected for six times into the HPLC system as per test procedure.

Acceptance criteria:

a.     All individual assays of sumatriptan tablets should be within 98 % - 102%.

b.     Relative standard deviation of % assay results should not be more than 2.0 %.

 

B) Method precision:

From sample and stock solution, six replicates of solutions of 25 ppm were prepared and injected into the HPLC system as per test procedure.

 

Acceptance criteria:

a.The all individual assays of sumatriptan tablets should be within 98 % - 102 %.

b. Relative standard deviation of % assay results should not be more than 2.0 %

 

Accuracy:

A series of solutions were prepared in triplicate (6 preparations for 50 and 150%level) by spiking the sumatriptan drug substance on placebo in the range of about 50 to 150 of test concentration and analysed as per specification level triplicate injections for 50%, 100% and 150%. Individual % recovery, mean % recovery, % RSD and linearity were calculated at each level.

 

Limit of detection (LOD):

Calibration curve was repeated for 3 times and the standard deviation (SD) of the intercepts was calculated. The LOD was determined by the formula:

                LOD = 3.3  σ /  S

Where σ = Standard deviation of Intercepts of calibration curves

 

S = Mean of slopes of the calibration curves

The slope S may be estimated from the calibration curve of the analyte.

 

Limit of quantification (LOQ):

Calibration curve was repeated for 3 times and the standard deviation (SD) of the intercepts was calculated The LOQ was determined by the formula:

LOQ = 10 σ /  S

 

Robustness:

Robustness was studied by testing the influence of small changes in pH of buffer (± 0.2 units), column temperature (± 5%), organic content of mobile phase (± 2%) and flow rate (± 5%).¹⁰

 

Degradation studies:

As per ICH guidelines, the forced degradation studies were performed on drug substance to establish its inherent stability characteristics in order to demonstrate selectivity and stability indicating capability of the proposed method. The standard substances were exposed to forced degradation studies such as oxidation, acid degradation, alkali degradation, dry heat degradation, photo stability and neutral degradation studies.

 

Acid degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add 2ml of 5% HCl, add little amount of diluents, heated at 80^oC for 20min. Cool and neutralize the solution by adding 2ml of 5% NaOH and the volume was adjusted with diluents. The above solutions were filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Acidic degradation blank was prepared in the same way without using sample.

 

Alkali degradation:

From the stock sample solution, 5ml of sample was transferred into 20ml volumetric flask and add 2ml of 5% NaOH, add little amount of diluents, heated at 80^oC for 20 min. Cool and neutralize the solution by adding 2ml of 5% HCl and made up to the volume with diluents. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Alkali degradation blank was prepared in the same way without using sample.

 

Peroxide degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add 2ml of H₂O₂, add little amount of diluents, heated at 80^oC for 20min. Cool and made up to the volume with diluents. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Peroxide degradation blank was prepared in the same way without using sample.

 

Reduction degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add 2ml of Sodium bisulphate, add little amount of diluents, heated at 80^oC for 20min. Cool and made up to the volume with diluents. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Reduction degradation blank was prepared in the same way without using sample.

 

Thermal degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add diluent, heated at 105^oC for 24 hrs. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Thermal degradation blank was prepared in the same way without using sample.

 

Photolytic degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add diluent, subjected to sun light for 24 hrs. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Photolytic degradation blank was prepared in the same way without using sample.

 

Humidity degradation:

From the sample stock solution 5ml of sample was transferred into 20ml volumetric flask and add little amount of diluent, subjected to 90% relative humidity at 25^oC for 24 hrs. The volume was made up to the mark with diluent. The above solution was filtered through 0.45µm nylon syringe filter by discarding first 4ml solution. Humidity degradation blank was prepared in the same way without using sample.

 

Assay:

Prior to the injection of drug solutions, the column was equilibrated for at least 30 min, with the mobile phase flowing through the system with a flow rate of 1.0 ml/min. Then 20 µl of prepared standard and sample solutions were injected for six times and 2 times respectively. The chromatograms were recorded to measure peak responses in sumatriptan standard and sample solutions.

 

RESULTS:

In the current study, the determination of sumatriptan was shown. Numerous proportions of buffer and solvents were assessed in order to obtain appropriate composition of the mobile phase. Innumerable experiments were accomplished by altering the concentration and pH of mobile phase, stationary phase selection etc., to optimize the chromatographic conditions to accomplish improved efficiency of the chromatographic system. The trial-1 chromatogram using water in the mobile phase produced a chromatogram with, high   retention time and peak was asymmetry, water was replaced with buffer (fig.1). The trial-2 chromatogram demonstrated broad peak. To overcome this pH was changed in the next trial (fig.2). The trial-3 chromatogram displayed low plate count and to get increase in theoretical plate, another trial was performed by changing pH (fig-3). The trial-4 chromatogram displayed good peak shape (fig-4). The trial-5 chromatogram (sumatriptan) eluted with good peak shape and symmetry. Retention time and tailing were within the limit (fig.5). The component was eluted at lower retention time with good reproducibility. Hence this method was optimized.

 

Fig. 1: First trial chromatogram run Optimized Chromatographic conditions

 

Fig.2: Second trial chromatogram run

 

Fig.3: Third trial chromatogram run

 

Fig.4: Fourth trial chromatogram run

 

System suitability:

System suitability results were well defined by several representative chromatographic trials. Retention time, %RSD, standard deviation, mean area, USP plate count, and tailing factor were evaluated by six replicate injections of drug (Sumatriptan). The results were shown in table.1.

 

Fig.5: Optimized chromatogram of Sumatriptan by proposed method

 

Linearity:

The linearity of the optimized method was determined for six concentrations and the correlation coefficient for sumatriptan was found to be 0.99998 (fig.6). The calibration curve was linear for 6 concentrations. Linearity was plotted between concentration and peak area. Slope and Y intercept was found to be 24908 and 277063. It shows that the linearity was within limit and obeys beer lambert’s law. The linearity results were listed in table 2.

 

Table 1: System suitability data of sumatriptan

 

Fig No: 6 Calibration curve showing Linearity graph of Sumatriptan.

 

 

 

 

Table 2: Table of linearity showing results for slope and intercept

 

Precision:

System precision (Repeatability):

From the system precision studies it was observed that % RSD of peak area was found to be 0.260. All the parameters were within the limit. The Precision results were displayed in table.3

 

Table No 3: Showing Precision observation of Sumatriptan

 

Accuracy:

A series of solutions were prepared in triplicate (6 preparations for 50 and 150%level) by spiking the sumatriptan drug substance on placebo in the range of about 50 to 150 of test concentration and analyzed as per specification level triplicate injections for 50%, 100% and 150%. Individual % recovery, mean % recovery, % RSD and linearity   were calculated at each   level. The results were revealed in table 4.

 

Table no 4: Showing Accuracy observation of Sumatriptan.

 

Limit of Detection and Limit of Quantification:

LOD and LOQ were determined by signal to noise ratio. The limit of detection and limit of quantification were found to be 1.967µg/ml and 5.961µg/ml which was within the acceptance range. The results were shown in table 5.

 

Table 5: Robustness Data

 

Table No 6: Data showing forced degradation studies of sumatriptan.

Forced degradation studies:

Degradation studies were performed with formulation and degraded samples. The % degradation of the drug for acid, alkali, peroxide, reduction, thermal, UV and humidity were found to be 5.34, 4.12, 4.66, 3.9, 2.19, 1.42, 1.32 respectively which was within the limit. Degradation data was mentioned in table.6

 

Assay of marketed formulation:

Standard and sample solution was injected separately into the system. The % RSD of marketed formulation was found to be 0.43. The results of assay (marketed formulation) were demonstrated in table.6

 

Table 7: Assay of formulation

 

DISCUSSION:

The development of an analytical method for the determination of drugs by HPLC has received considerable attention in recent years because of their importance in quality control of drugs and drug products. Linearity is the method ability to obtain test results, which are directly proportional to the concentration of analyte in the sample. Sumatriptan showed a linearity response curve. The correlation coefficient was found to be 0.99998, hence it was greater than 0.999 which was within the limit. There is a greater correlation between peak area and concentration of analyte. The slope and Y-Intercept was found to be 24908 and 277063. The method of precision determines the closeness of agreement between a series of measurements of the same sample. From the system precision studies it was observed that % RSD of peak area was found to be 0.260. All the parameters were within the limit. The values obtained were NMT 2.0% which was within the limit and good precision of assay method was confirmed. To demonstrate the accuracy of the method, standard addition and recovery experiments were conducted. The measure of method capacity to remain unaffected by small, but deliberate changes in chromatographic conditions known as robustness. Retention time and tailing factor were shown adherence to the limits. The limit of detection is the lowest concentration of analyte that can be detected. The limit of quantification is the lowest concentration of analyte that can be quantified. The LOD and LOQ determined by signal to noise ratio approach as defined in ICH. The LOD and LOQ were found to be 1.967μg/ml and 5.961μg/ml respectively, which was within the acceptance range. The purity threshold was found to be more than the purity angle which was within the acceptable range. This shows that the method was specific and stability indicating. Validation of the developed method was done as per the ICH guidelines. The proposed method was found to be simple, precise, accurate and validated according to ICH guidelines with respect to linearity, precision, accuracy, robustness and specificity studies which remained well within the limit.

 

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Accepted on 02.01.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5856-5862.