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INTRODUCTION
Migraine is considered one of the most frequent and most severe types of primary headaches with significant impairment of the patient’s functioning. A significant fraction of the world population is affected by the Migraine syndrome due to a stressful lifestyle. This disorder occurs in approximately 11.6% populations of the world. As compared to men, women are mostly affected by this disease (Lade et al., 2022). Almotriptan Malate is one of the many Triptans-a group of drugs that are usually recommended for the acute management of migraines. Migraine is thought to be a disease of the serotonin receptors in the brain and these drugs affect these receptors to help provide relief (Kalra and Elliott, 2007).
Almotriptan Malate (Figure 1) is a selective 5-Hydroxytryptamine (5-HT) receptor agonist used in the acute treatment of migraine. Several analytical methods have been described for the estimation of Almotriptan in pharmaceutical dosage forms and biological fluids employing LC-MS/MS (Ravikumar et al., 2012). Almotriptan is the first FDA-approved drug to treat migraine in adults with or without an aura. Almotriptan Malate is characterized by a short half-life and up to 3 hr; The bioavailability of the oral form amounted to 69.1%. The currently available oral delivery system of Almotriptan Malate has limited bioavailability because of poor aqueous solubility (Lade et al., 2022).
Niosomes are also known as Non-ionic Surfactant Vesicles (NSV) is nanoscale-sized vesicles that have an aqueous-filled core and one or several lipid layers (Makeshwar and Wasankar, 2013). The advantages of niosomes include very low toxicity, stable chemical structures, biocompatibility, ability to encapsulate hydrophilic or lipophilic drugs, and low cost of this formulation (Patel et al., 2014). Poloxamer 407 is commonly used to prepare thermosensitive in situ gel to increase the viscosity and mucoadhesiveness of the formulations. In some investigations, temperature-sensitive gelling systems have been employed for the nasal administration of drugs. This in situ gel converts gel at systemic temperature (31-33ºC) and remains in liquid condition at room temperature (25±0.56) (Ravikrishna and Krishnaveni, 2019).
The International Council on Harmonization (ICH) requirements for analytical method validation are followed by this method, which seeks to be dependable, quick, and economical. In the present study, we report the development and validation of a UV-visible spectrophotometric method for the estimation of Almotriptan malate in bulk and AM-loaded Niosomes (Suneetha et al., 2012). The comparison studies were done with the previous studies, and some limitations were found and reported in Table 1.
The validation parameters as per ICH guidelines are Accuracy, Precision, Repeatability, Intermediate Precision, Reproducibility, Limit of Detection (LOD), Limit of Quantitation (LOQ), Range and Linearity, and Solvent Stability (Sharma et al., 2018).
MATERIALS AND METHODS
Drug sample
Almotriptan Malate (pure Drug) was procured from MSN Laboratories Ltd., Pvt. Hyderabad.
Reagents and chemicals
The experiment’s analytical-grade chemicals and reagents were obtained from the KLE College of Pharmacy’s storage facility in Belagavi. Potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ethanol of HPLC grade were used.
Instruments and Apparatus
The Shimadzu UV/visible spectrophotometer, model 1800, equipped with UV-Probe software, was used in conjunction with 10 mm Quartz cuvettes and automated wavelength adjustment. Before use, pipettes, volumetric flasks, and measuring cylinders used in the laboratory were calibrated.
Method Development
The first step considered while developing the UV spectrophotometric technique included: the selection of the appropriate solvent system and determination of the maximum absorbance wavelength (λmax). After a literature review in the area of solubility, Almotriptan malate solubility studies were conducted in a real-life environment. Numerous experiments in a range of solvents (methanol, water, ethanol, Phosphate buffer pH 6.4, and Phosphate buffer 6.8) were conducted to choose the mobile phase. Finally, a 50:50% v/v mixture of solvents including ethanol and phosphate buffer pH 6.8 was determined. Solutions containing analytes had their UV spectra examined in the 200-400 nm range.
Method Validation
The analytical technique was tested for performance validation by assessing its linearity, accuracy, precision, robustness solvent stability, specificity, and ruggedness under the ICH criteria for analytical method validation. The method’s appropriateness was confirmed by using ICH guidelines Q2A and Q2B 12 (ICH guidance Q2a; ICH guidance Q2b).
Preparation of Solvent System
Mobile Phase
50 mL of precisely measured ethanol was dissolved in 50 mL of freshly prepared phosphate buffer pH 6.8.
Standard Stock Solution
The 10 mL volumetric flask containing 10 mg of Almotriptan malate was used to prepare the standard stock solution. Almotriptan Malate was weighed and combined with 50% v/v ethanol: 50%v/v phosphate buffer pH 6.8 to develop a stock solution that contained 1000 µg/mL.
Working stock solutions
From the 0.1 mL Almotriptan Malate standard stock solution, 1 mL was pipette into 10 mLvolumetric flask, the volume was made up with mobile phase before the analysis. Almotriptan Malate concentrations ranging from 0.2 to 1 mL were taken out and pipetted into identical 10 mL volumetric flasks. Using the same mobile phase, the volume was adjusted to the appropriate level.
Selection of wavelength
Almotriptan Malate was tested at 400-200 nm using 10 µg/mL of the corresponding stock solution as ethanol: pH 6.8 phosphate buffer blank solutions (50:50% v/v).
Linearity and range
The Almotriptan Malate was mixed in varied amounts (0.2-1 mL for Almotriptan Malate) to achieve different concentrations (1-5 µg/mL) and then scanned. To accommodate for variability, this process was carried out three times, statistical analysis (regression) was used to see whether changes in Almotriptan Malate concentration could be used to predict changes in absorbance (Mishra and Mundada, 2021).
Limit of Detection and Limit of Quantification
The ICH defines the Quantitation Limit (QL), also referred to as the limit of quantification, as LOQ=10σ/S, as well as the detection limit (DL or LOD) as LOQ=3.3σ/S. S stands for the calibration curve’s slope, and σ for the response’s standard deviation (Sanchez, 2018; Srinivas et al., 2021).
| Journal name | Title | Limitations |
|---|---|---|
| International Journal of Medicinal Chemistry and Analysis. | Spectrophotometric estimation of Almotriptan malate in bulk and pharmaceutical formulations by multivariate technique (Prabhavthi et al., 2016). | The paper mentions that there is no interference observed from excipients of the formulation, but it is not clear about the excipients that have been tested or what conditions the validation of this section of the method has been done. The phenomenon of long-term stability of the analyzed samples is not considered in the study. |
| World Journal of Pharmaceutical Research. | A simple spectrophotometric assay of Almotriptan malate in bulk and pharmaceutical formulations (Prasad et al., 2012). | The authors do not specify what range of concentrations the method is effective and could therefore be an issue for medicines that have varying levels of Almotriptan malate. However, the employing of chloroform in the method may take some precautionary issues for safety and environmental concerns hence not applicable to some laboratories. |
| International Journal of Scientific and Technology Research | Visible spectrophotometric analysis of Almotriptan Malate in bulk and formulations (Kumari et al.,2012). | Previous methods were found to be both tedious and less specific, highlighting the necessity for improvement in the analytical techniques employed. Conventional visible spectrophotometric methods used in the determination of Almotriptan malate may still cause problems in terms of specificity. |
| International Journal Chemical Sciences. | Spectrophotometric determination of a Almotriptan Malate in pharmaceutical dosage forms (Jesus et al., 2020). | The research did not provide adequate information on the conditions studied for the formation of colored complexes and might cause some difficulties in the reproduction of the results. The research did not attempt to establish the efficiency of the method or the stability of the coloured complex. |
Precision
The study developed the interday and intraday precision of the Almotriptan Malate by executing 3 replicates of concentrations: 0.2 mL, 0.6 mL, and 1 mL of Almotriptan Malate at the 227 λmax. Almotriptan Malate percentage RSD values were developed (Patel et al., 2023).
Ruggedness
Ruggedness is the ability of the approach to tolerate both intentional and inadvertent changes. Triplicate measurements were taken at three distinct concentrations using two separate UV equipment (Gaikwad et al., 2023).
Robustness
To evaluate the robustness (reliability) of the UV spectrophotometry approach, two distinct wavelengths of 227 nm±2 nm were used to analyze the drug. This produced equations for multiple regression and their associated coefficients, which were useful in assessing how well the procedure worked in slightly different circumstances (Friedrich et al., 2009).
Accuracy (recovery studies)
A recovery study was carried out to evaluate the accuracy of the method. This was developed by pre-analysing a sample and then adding standard Almotriptan Malate solution at three distinct concentration levels (50%, 100%, and 150%) (Jain et al., 2011).
Solvent and standard stock solution stability
For three days (72 hr), the solutions were stored in unstable circumstances to test the experiment’s stability. Following this time, they assessed if the solutions had not changed (Uday et al., 2021).
Formulation of Niosomes
Almotriptan Malate-loaded Niosomes were prepared by an ethanol injection method. Emulsifier, Lipid, and Almotriptan Malate were accurately weighed and dissolved into 10mL of solvent using bath sonication at 60ºC. At the same temperature, the clear organic solution was very rapidly poured into the solution of the aqueous phase and the mixture was vigorously stirred at 500-650 rpm by the Teflon-coated bead in a Remi magnetic stirrer. The liquids turned into a milky solution as a confirmation that Niosomes were being formed within the solution. To evaporate the solvent the solution was allowed to evaporate while stirring for 30 min at room temperature, and the samples were placed in vacuum for 10-15 min. Last the volume of the Niosomal dispersion was, further diluted up to 30 mL by using water. The obtained Niosomal dispersion was then sonicated under a probe sonicator and filtered through 2-20 m filter out to have the homogenized size distribution and proper vesicle closure (Katrolia et al., 2019; Estupiñan et al., 2020).
| Sl. No. | Parameters | Specifications |
|---|---|---|
| 1 | Analytes | Almotriptan Malate |
| 2 | Solvent | Ethanol: Phosphate buffer pH 6.8 (50:50% v/v) |
| 3 | Maximum absorbance of Almotriptan Malate | 227nm |
| LOD and LOQ Data | |
|---|---|
| Analytes | Almotriptan Malate Concentration (µg/mL) |
| LOD | 1.12 µg/mL |
| LOQ | 3.42 µg/mL |
Characterization of Niosomal Dispersion
Drug-loaded Niosomal Dispersion was diluted with distilled water to achieve appropriate concentrations for analysis. Dynamic Light Scattering (DLS) using a Malvern Zeta Sizer instrument was employed to determine the Polydispersity Index (PDI) and zeta potential of the drug-loaded Niosomal Dispersion. All measurements were made in triplicate to minimize the probability of data variability.
Separation of Unentrapped Drug from Niosomes
A volume of 2.5 mL of drug-loaded Niosomal dispersion was subjected to centrifugation to isolate the unencapsulated drug fraction. This process was performed using a KUBOTA-7000 high-capacity refrigerated centrifuge (Japan) at 15,000 rpm for 30 m at 4ºC. Following centrifugation, 0.1 mL of the supernatant, containing the free (unentrapped) drug, was collected, and subsequently diluted in 10 mL of Milli-Q water. The diluted supernatant was filtered through a Millex® Syringe Filter (PTFE) to eliminate Niosomal particles.
Quantification of Encapsulated Drug UV Spectrophotometrically
The Almotriptan Malate-loaded Niosomal dispersion sample was again centrifuged using a High-Speed Refrigerated Centrifuge (Floor Model, 7000 Kubota, Japan) at 19,000 rpm for 60 min. Almotriptan Malate content in the Niosomes was calculated based on the absorbance of the appropriately diluted supernatant at 227 nm using UV/visible spectroscopy.
The % EE was calculated to be;
RESULTS
Method development
To perform estimation, the analyte must dissolve in a solvent. Method development parameters are illustrated in Table 2.
Method Validation
Almotriptan Malate showed maximum absorption at 227 nm, respectively shown in Figure 2.
Linearity
The experiment showed a linear relationship between Almotriptan Malate concentration and absorbance of 0.2-1 μg/mL (Figure 3). This was valid at the examined wavelength of 227 nm. Strong correlation coefficients (R²) were found to support the outcome, with Almotriptan malate having an R2 of 0.9974.
LOD and LOQ
The study assessed the Limit of Detection (LOD) and Limit of Quantitation (LOQ), two crucial criteria for our analytical approach. The lowest amount of drug (analyte) that we can accurately measure but still reliably detect is known as the limit of detection, or LOD. On the other hand, LOQ is defined as the limit below which the presence of the medication cannot be confirmed accurately and measured. Table 3 contains the precise LOD and LOQ values.
| Concentration (µg/mL) | Absorbance (Average of three replicates) |
|---|---|
| 0.6 | 0.611 |
| 0.6 | 0.619 |
| 0.6 | 0.612 |
| 0.6 | 0.612 |
| 0.6 | 0.613 |
| 0.6 | 0.611 |
| STD Dev | 0.003 |
| Average | 0.613 |
| %RSD | 0.494 |
Precision
The percent Relative Standard Deviation (%RSD), estimated for three replicates at three distinct concentrations for Almotriptan Malate, consistently was found below 2%, indicating that the analysis procedure was accurate. Tables 4-6 give the detailed results.
Ruggedness
The %RSD for Almotriptan Malate was measured using various analysts and instruments to evaluate the robustness of the method. All replicates’ %RSD values stayed below 2%, demonstrating the method’s reliability and repeatability. Table 7 provides more information on these results.
Robustness
The analysis procedure was slightly altered to evaluate robustness. All of the %RSD values that were determined using the regression coefficients (the resultant data) were less than 2%. These outcomes, shown in Tables 8 and 9 show that little modifications do not affect the procedure.
| Concentration (µg/mL) | Almotriptan Malate (227 nm) | ||
|---|---|---|---|
| 0th hr | 1st hr | 5th hr | |
| 0.2 | 0.235 | 0.236 | 0.232 |
| %RSD | 0.888 | 0.651 | 0.863 |
| 0.6 | 0.612 | 0.619 | 0.615 |
| %RSD | 0.57 | 0.161 | 0.324 |
| 1 | 0.983 | 0.98 | 0.985 |
| %RSD | 0.256 | 0.41 | 0.203 |
| Concentration(µg/mL) | Almotriptan Malate (227 nm) | ||
|---|---|---|---|
| Day 1 | Day 2 | Day 3 | |
| Almotriptan Malate | Absorbance | Absorbance | Absorbance |
| 0.2 | 0.236 | 0.235 | 0.232 |
| %RSD | 0.888 | 0.843 | 0.888 |
| 0.6 | 0.612 | 0.615 | 0.619 |
| %RSD | 0.57 | 0.337 | 0.487 |
| 1 | 0.988 | 0.985 | 0.99 |
| %RSD | 0.254 | 0.309 | 0.266 |
| Concentration (µg/mL) | Almotriptan Malate (227 nm) | |
|---|---|---|
| Change in Analyst | Change in Instrument | |
| Almotriptan Malate | Absorbance | Absorbance |
| 0.2 | 0.232 | 0.233 |
| %RSD | 0.893 | 0.851 |
| 0.6 | 0.62 | 0.619 |
| %RSD | 0.817 | 0.404 |
| 1 | 0.99 | 0.989 |
| %RSD | 0.254 | 0.154 |
Accuracy (recovery studies)
Almotriptan Malate Samples were spiked and recovery was measured to test accuracy. Almotriptan Malate recovered exceptionally well (93.3-96.7%); see Table 10.
Solvent and standard stock solution stability
To prove that the working solution remains stable during analysis and does not deteriorate in the diluent media, utilizing regression coefficients, the %RSD values were all less than 2%. Table 11 results show that the stock solutions have undergone only minor alterations, indicating that they have remained stable throughout the study.
Characterization of Niosomal Dispersion
The particle size distribution was found at 148.3 (Figure 4A) nm indicating a desirable size range for the Niosomes. A Polydispersity Index (PDI) of 0.207, close to 1, suggested a uniform particle size distribution. The zeta potential of -29.07 mV (Figure 4B) showed good stability.
Spectrophotometric Analysis of Niosomes
Analysis of the final Niosomal dispersion revealed high Entrapment Efficiencies (EE) of 82.8% for Almotriptan Malate respectively. These values suggest the desirable entrapment efficiency of a major portion of the drug within the Niosomes.
DISCUSSION
The absorptivity of the drugs remained consistent across a concentration range of 0.2-1 μg/mL for Almotriptan Malate. This behaviour complies with beer-lambert’s law where there should be a linear response which means that the process used can be trusted to analyze the drug within the ranges being tested. Very high R² values specifically indicate that there exists a very good linear relationship between concentration and absorbance spot for the drug. From the results, it can be concluded, that LOQ is higher than LOD because it demands greater sensitivity and precision from the analysis. The test was good in terms of accuracy since the results were similar for all degrees of the drug being analysed. The robustness of the method can be observed from the fact that it gives fairly comparable results for somewhat different conditions in the laboratory or analyses. The Results at different Almotriptan Malate concentration levels showed %RSD values below 2% showing that the present method is precise and reproducible. The robustness of a method is indicated by its reliability in routine use. This entailed consciously using variation in the analysis method and comparing the resultant effect. The method’s resistance to minor changes (as shown in the results) makes it reliable for routine analysis. The accuracy of the method, or how closely it reflects the true amount of drug present, was evaluated through a recovery study. Solution stability studies ensured that the working solution was not degraded hence the results of the final assay were not tainted by any false results. This stability is so important in order not to affect the outcome. Niosomes efficiently captured a large portion of the drugs: 59.8 for Almotriptan Malate (high entrapment efficiency). This finding suggests a promising approach for targeted delivery applications, where the drugs can be delivered to specific sites within the body.
| Change in wavelength | |||
|---|---|---|---|
| Concentration (µg/mL) | Almotriptan Malate (225 nm) | Almotriptan Malate(227 nm) | Almotriptan Malate(229 nm) |
| Almotriptan Malate | Absorbance | Absorbance | Absorbance |
| 0.2 | 0.221 | 0.236 | 0.231 |
| %RSD | 0.896 | 0.888 | 0.858 |
| 0.6 | 0.609 | 0.612 | 0.619 |
| %RSD | 0.51 | 0.57 | 0.57 |
| 1 | 0.978 | 0.988 | 0.989 |
| %RSD | 0.256 | 0.254 | 0.21 |
| Concentration | Methanol: Buffer (52:48) | Methanol: Buffer (48:52) |
|---|---|---|
| 0.6 | 0.622 | 0.615 |
| %RSD | 0.729 | 0.53106 |
| Level | Concentration (mg/L) | Absorbance | Mean Absorbance | Measured Concentration (mg/L) | %Recovery |
|---|---|---|---|---|---|
| 50% | 0.3 | 0.562 | 0.559 | 0.28 | 93.3% |
| 0.554 | |||||
| 0.561 | |||||
| 100% | 0.6 | 0.745 | 0.74 | 0.58 | 96.7% |
| 0.735 | |||||
| 0.74 | |||||
| 150% | 0.9 | 0.91 | 0.909 | 0.85 | 94.4% |
| 0.915 | |||||
| 0.901 |
| Concentration (µg/mL) | Almotriptan Malate (227 nm) | |
|---|---|---|
| Old | Fresh | |
| Absorbance | Absorbance | |
| 0.2 | 0.235 | 0.231 |
| %RSD | 0.888 | 0.893 |
| 0.6 | 0.612 | 0.61 |
| %RSD | 0.57 | 0.817 |
| 1 | 0.983 | 0.985 |
| %RSD | 0.256 | 0.254 |
CONCLUSION
The method for the simultaneous estimation of Almotriptan Malate was developed and validated according to ICH guidelines. The statistical analysis results confirmed the simplicity, specificity, selectivity, linearity, precision, ruggedness, robustness, Accuracy (recovery studies), Solvent and standard stock solution stability, Characterization of Niosomal Dispersion, and reproducibility of the developed method for simultaneous estimating Almotriptan Malate in bulk and AM-loaded Niosomes.
The ICH recommendations were followed in the development and validation of a technique for the simultaneous determination of Almotriptan Malate. The statistical analysis results demonstrated that the existing approach is straightforward, robust, reproducible, specific, linear, and selective for the simultaneous quantification of Almotriptan malate in bulk and pharmaceutical formulation.