INTRODUCTION:

One of the most common and serious health disorders associated with the mental well-being of a person is depression which is a serious condition characterized by persistent sadness for at least 2 weeks. According to World Health Organisation (WHO), approximately 280 million people suffer from depression worldwide.¹(1S-cis)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthalenamine², Sertraline (SER) has proved to be one of the most effective drugs used in the treatment of depression, It binds with 5-HT1A receptor and produces antidepressant action by inhibiting the reuptake of SER in pre-synaptic neurons. SSRIs don’t have any significant effect on norepinephrine transporter (NET), serotonergic, dopaminergic, histaminergic, and cholinergic receptors.^3,4

WHO has included SER in the list of essential medicines, as a treatment for post-traumatic stress disorder (PTSD), major depressive disorder, obsessive–compulsive disorder (OCD) in children, panic attacks and social anxiety disorders.^5,6Accurate and reliable calculation of SER is crucial for conducting bioequivalent studies and guaranteeing the quality control of pharmaceutical formulations. Evaluating a wide range of chemical ingredient classes is possible by utilizing several chromatographic techniques quantitatively, which have the advantages of quick development, high precision, accuracy, specificity, and sensitivity. To minimize time- for consistency, optimization, validation, and instrumentation, the methodology should be set early in the creation of analytical procedures, aligned with the purpose and scope of the technique.⁷While developing a technique, many parameters, including linearity, specificity, accuracy, limits of quantitation (LOQ), limits of detection (LOD), should be taken into consideration. Suitability for producing consistent findings is confirmed via their validation. ICH) offers conclusive advice on the criteria for validating analytical procedures.⁸SER and its derivatives have been determined using a variety of techniques, such as UV spectroscopy, high-performance liquid chromatography (HPLC), and high-performance thin-layer chromatography (HPTLC). However, there are limited data on SER estimation using RP-HPLC in pharmaceutical formulations. Therefore, the primary objective of this study was to create a unique, simple, precise, and sensitive RP-HPLC technique for SER quantification in pharmaceutical formulations containing both active pharmaceutical ingredients (API) and SER nanofibers. To verify the reliability, repeatability, and sensitivity, the devised analytical technique was carefully verified and statistically evaluated for a variety of parameters in accordance with ICH recommendations. Due to its sensitivity and high throughput capabilities, HPLC has been one of the analytical methods that are most often employed for the analysis of pharmaceuticals.⁹Reverse-phase high-performance liquid chromatography (RP-HPLC) is a frequently used method for the separation and quantification of hydrophobic compounds, including SER. The purpose of this work was to develop and validate the RP-HPLC technique for SER estimation in nanofibers formulation. The developed approach will provide a precise, accurate, and reliable way to determine SER in pharmaceutical formulations, allowing quality control analyses and supporting bioequivalence investigations.¹⁰

Figure 1. Chemical structure of sertraline

MATERIALS AND METHODS:

Materials:

The gift sample of sertraline SER (API) was procured from Abbott India Ltd., Goa, India. Eudragit L100-55 from Evonik Industries AG, Germany), Parteck SLC 500 from Merck, Germany, and Phosphoric acid was purchased from Sigma-Aldrich. Spectro chem. Pvt. Ltd, located in Mumbai, India, supplied the acetonitrile of HPLC grade on request. Weighing tasks were performed using a digital electronic balance with model number BL-220H, manufactured by Contench and Citizen, Mumbai, India.

Methods:

Determination of absorption maxima:

The UV-visible spectrophotometer was used to scan SER solution having a concentration of (10 μg /mL in methanol) within the UV wavelength range. The λ_maxso measured was compared with the values reported in the literature.

Equipment and chromatographic conditions:

Analysis was conducted by using RP-HPLC (1100 Infinity LC, Agilent technologies) method, using mobile phase with phosphoric acid (0.5%) and acetonitrile in the ratio of (45: 55) (%v/v), Inertsil ODS 3V C18 column (250.0 ˟ 4.6 mm, 5.0μm, Agilent) at flow rate: 1.0 mL/min, column temperature 30 ℃ with injection volume 30 μL and diode Array Detector (DAD) at λ_max 274nm with run time 5 min and retention time 3.8 min.

Sertraline-loaded nanofibers preparation:

The electrospinning method using various polymers was used to fabricate nanofibers where the polymers act as drug carriers. The polymers were solubilised in an organic solvent blend in suitable ratios. Electrospinning solutions were obtained by dissolving the polymers in a mixture of solvents using a magnetic stirrer at room temperature for 24 hrs. Subsequently, SER was solublised in the blend of organic solvents and added to the polymeric solutions. The solutions so prepared were loaded in a 20 mL plastic syringe and pumped through the syringe fixed at the metal spinneret support. The electric field was applied by using a high voltage between the syringe needle which acts as the primary electrode and the collector which act as the counter electrode ultimately the dry fibers were collected on a metallic plate.^11,12

Preparation of standard solution:

To make SER stock solutions, 10mg of the SER was dissolved in a mixture containing a ratio of (90:10 v/v) solution containing 90mL of acetonitrile and water to obtain a solution having concentration of 1000μg/mL. To completely dissolve the SER the solution so obtained was immersed for 5 min in an ultrasonic bath.

Preparation of working standard solution:

Solution of concentrations of 20, 40, 60, 80,100, and 120μg/mL were prepared using 1000 μg/mL stock solution. Before doing the analysis all the solutions were filtered by using a 45 μm membrane.

Preparation of sample solution:

About 4 mL of the supernatant liquid was taken to prepare the sample. A mobile phase consisting of 90% acetonitrile and 10% water was used to adjust the volume to 10 mL. The sample was then subjected to sonication in an ultrasonic bath for 5 min. The sample was ultimately filtered before analysis using a 45 μm membrane filter.

Validation of method:

The ICH guideline was followed to conduct the validation of the analytical methodology, various parameters were validated including specificity, linearity, detection limit (LOD), quantification limit (LOQ), precision, accuracy, robustness, and ruggedness.^{13, 14}

Specificity:

Excipients interference in SER determination was assessed to gauge the method's specificity. A comparison of SER nanofibers and without the drug (Blank) was carried out by comparing the chromatograms of the two formulations to confirm the excipient's interference with drug measurement.¹⁵

Linearity:

SER standard solutions having concentrations of 20, 40, 60, 80, 100, and 120 μg/mL were taken. The calibration curve was created, by plotting a graph of concentration against the corresponding peak area, further, the least square regression approach was used to determine the linearity, and linear regression was used to determine the line's equation. Calculations were used to determine the respective values of the slope, Y-intercept, and correlation coefficient of the regression line.¹⁶

LOD and LOQ determination:

Detection limits (LOD) and quantification limits (LOQ) were used to determine the sensitivity of the method. The LOD is defined as the minimum concentration of the analyte that consistently triggers a reaction, yet may not be precisely quantifiable every time. On the other hand, the LOQ corresponds to the minimum analyte concentration capable of yielding an accurate result. LOD and LOQ were calculated by using the below-mentioned formula with the help of standard deviation of response (SD) and slope (S) methods. For each study, a minimum of three separate analyses were done. ¹⁷

LOD = 3.3 σ /S ; LOQ= 10 σ /S

σ = the standard deviation of the response

S = the slope of the calibration curve

Precision:

The precision of the procedure was evaluated by executing intra-day (repeatability) and inter-day (intermediate) analyses using triplicate injections of the SER, the solution was examined three times on the same day (Day 1, n = 3) to accomplish the intra-day precision, while inter-day precision was performed by examining these solutions for three times on three different days. The relative standard deviation (%RSD) of a set of measurements was used to express the precision data.¹⁸

Accuracy:

The evaluation of the procedure for checking accuracy (recovery) was done by comparing the samples having known drug concentration in the blank sample of nanofiber with the known concentrations of SER standard solutions which were added to a blank nanofiber solution to generate concentrations of 5, 10, and 15μg/mL. The preparation of each sample was done in triplicate, and the recovery rate was assessed.¹⁹

Ruggedness:

Evaluation of the method's ruggedness was carried out by doing subtle and intentional changes in the chromatographic conditions. The effects of various parameters like change in the flow rate, column temperature, and percentage of organic solvent on retention time and SER content percentage were studied and evaluated.^20,21

RESULTS AND DISCUSSION:

The approach that was developed thus underwent ICH-recommended validation. Validation was done to confirm that safe and valid drug products are administered to the patients for achieving maximum therapeutic benefits and minimization of potential side effects.

Determination of Absorption Maxima:

The 10 μg/mL concentration of SER solution was scanned by using a UV spectrophotometer. The recorded spectrum showed the absorbance maxima (λ_max) at 274 nm as shown in Figure. 2.

Figure 2. λ_maxfor sertraline in methanol

Method Validation:

Specificity:

The method's specificity was determined by testing its ability to identify the drug in a mixture with other formulation excipients. Figure 3 shows the chromatogram of SER, Figure 4 shows the chromatogram of blank Nanofiber solution (placebo), and Figure 5 shows the chromatograms for SER nanofiber samples. The specificity analyses adequately proved there was no interference from any other excipients because none of the peaks displayed the same RT of SER. As a result, the perfect separation of SER from nanofibers justified the specificity and demonstrated that there was no interaction with the quantitative determination of SER from nanofiber formulation.

Figure 3. Chromatogram of SER (100 μg/mL)

Figure 4. Chromatogram obtained from Blank Nanofiber solution (placebo)

Figure 5. Chromatogram obtained from SER nanofiber formulation

Linearity:

The method was found to be linear in the range of 20-120 μg/mL for SE. The linearity determination coefficient (R²) for sertraline was found to be 0.9991.

Figure 6. Linearity curve of SER at 274 nm

Table1. Result of sertraline regression analysis

Parameter	Result
λ_max	274 nm
Beers law limit (μg/mL)	20-100
Regression Equation Data
Slope	4.5
Intercept	6.2
Correlation coefficient	0.9991

Accuracy:

The % recoveries of the findings show that the recoveries are well within the acceptability range (%RSD<2), indicating that the procedure is accurate according to the established standards (ICH, 2005).

Table 2. Results of statistical analysis of accuracy

Concentration level (%)	Pre-analyzed sample solution (μg/ml)	Amount of drug added (μg/ml)	% Recovery Mean ± SD	% RSD
50	10	5	99.6 ± 0.57	0.6
100	10	10	100.2 ± 1.11	1.1
150	10	15	102.4 ± 0.08	0.1

Precision:

Table 3 below provides the %RSD for intra-day and inter-day variation. The %RSD readings were found to be substantially within the 2% limit in both situations, proving the repeatability of the current method.

Table 3. Results of statistical analysis of intra-day assay and inter-day assay

Sr. No.	Concentration (μg/mL)	Intra-day precision		Inter-day precision
Sr. No.	Concentration (μg/mL)	Peak Area	Peak RT	Peak Area	Peak RT
1	100	522.77118	3.735	511.49854	3.857
2	100	523.23145	3.737	512.67987	3.861
3	100	523.47321	3.734	513.20697	3.867
4	100	523.22852	3.739	512.54706	3.87
5	100	522.37079	3.735	511.65564	3.871
6	100	522.55566	3.732	511.56763	3.871
Mean		522.9384683	3.735333333	512.1926183	3.866167
SD		0.4	0.0	0.7	0.0
%RSD		0.1	0.1	0.1	0.2

Values are mean±SD, n=3

Table4. Results of statistical analysis of ruggedness

Parameter		Quantity of Sertraline added (μg/mL)	Quantity of Sertraline detected (μg/mL)	% RSD
Change in the mobile phase	Buffer (0.5 % Phosphoric acid): Acetonitrile (45:55) (%v/v)	100	100.15 ± 0.19	0.2
	Buffer (0.5 % Phosphoric acid): Acetonitrile (50:50%)(%v/v)	100	100.42 ± 0.39	0.4
	Buffer (0.5 % Phosphoric acid): Acetonitrile (40: 60) (%v/v)	100	100.05 ± 0.29	0.3
Change in column temperature	25 ℃	100	99.86 ± 0.55	0.6
	30 ℃	100	100.37 ± 0.36	0.4
	35 ℃	100	99.88 ± 0.40	0.4
Change in flow rate	0.9 mL/min	100	99.62 ± 0.46	0.5
	1.0 mL/min	100	100.09 ± 0.69	0.7
	1.1 mL/min	100	99.67 ± 0.48	0.5

LOD and LOQ:

To determine the sensitivity of the method, LOD and LOQ were evaluated. The LOD for SER was found to be 2.71 μg/mL. On the other hand, the LOQ for SER was found to be 9.03 μg/mL. As per the acquired results, the developed method is sufficiently sensitive to estimate the SER.

Ruggedness:

Minor changes are made on purpose to test the method's robustness and ruggedness. The outcomes showed that under specific chromatographic circumstances, no obstruction affected drug findings. Peak areas and retention times showed no discernible change. Additionally, the drug content of sertraline was close to 100% for all evaluated situations, with an RSD of less than 2%. The collected findings demonstrated the suggested method's robustness and ruggedness under slight variations in chromatographic settings. (Table-4).

CONCLUSION:

The HPLC method was successfully developed for the quantitative determination of SER from bulk and nanofiber compositions. The validated method has shown exceptional specificity, linearity, accuracy, precision, and sensitivity to measure SER in nanofiber formulation. The simplicity of this method may be seen in its rapid evaluation times, use of an isocratic mobile phase elution mode, and accomplishment of clear resolution with low limits of detection (LOD) as well as quantification (LOQ) values. The suggested method was developed and validated in accordance with the requirements outlined by the ICH, making it ideal for routine SER analysis from a medicinal dosage formulation.

ABBREVIATIONS:

Selective serotonin reuptake inhibitor, (SSRI); Sertraline, (SER); Ultraviolet spectrophotometer, (UV); International Council of Harmonization, (ICH); Active pharmaceutical ingredient, (API); %Relative standard deviation, (%RSD); Standard deviation, (SD); Limits of detection, (LOD), Limits of quantitation, (LOQ), Correlation coefficient; (R²).

ACKNOWLEDGMENT:

The authors appreciate Bharati Vidyapeeth College of Pharmacy, Kolhapur, and School of Nanoscience and Technology, Kolhapur for providing the necessary facilities for carrying out research work.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 23.06.2023 Modified on 18.07.2023

Research J. Pharm. and Tech 2023; 16(8):3743-3748.

DOI: 10.52711/0974-360X.2023.00618