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 (Ladeet 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 (Ravikumaret 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 (Ladeet 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 (Patelet 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 (Suneethaet 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 (Sharmaet al., 2018).

Almotriptan Malate (pure Drug) was procured from MSN Laboratories Ltd., Pvt. Hyderabad.

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.

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.

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 (λ_max). 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.

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).

50 mL of precisely measured ethanol was dissolved in 50 mL of freshly prepared phosphate buffer pH 6.8.

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.

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.

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).

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).

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; Srinivaset al., 2021).

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 λ_max. Almotriptan Malate percentage RSD values were developed (Patelet al., 2023).

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 (Gaikwadet al., 2023).

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 (Friedrichet al., 2009).

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%) (Jainet al., 2011).

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 (Udayet al., 2021).

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 (Katroliaet al., 2019; Estupiñanet al., 2020).

Figure 1:
Structure of Almotriptan Malate.

Figure 2:
UV-spectrum of Almotriptan Malate.

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.

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.

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;

To perform estimation, the analyte must dissolve in a solvent. Method development parameters are illustrated in Table 2.

Almotriptan Malate showed maximum absorption at 227 nm, respectively shown in Figure 2.

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.

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.

Figure 3:
Calibration Curve.

Figure 4A:
Particle size.

Figure 4B:
Zeta potential.

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.

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.

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.

Almotriptan Malate Samples were spiked and recovery was measured to test accuracy. Almotriptan Malate recovered exceptionally well (93.3-96.7%); see Table 10.

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.

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.

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.

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.

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.

The authors are thankful to the Principal and management of KLE College of Pharmacy, Belagavi for providing the necessary facilities to conduct the work.