The use of QbD for the development and validation of a stability-indicating RP-HPLC method for the quantitation of nevirapine in bulk, tablet and niosome formulations

Introduction: A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the quantitation of nevirapine (NVP) in bulk drug, commercial tablets, and niosome formulations using an Analytical Quality by Design (AQbD) approach.

Methods: Critical analytical attributes and method parameters were identified and optimized using a Central Composite Design (CCD), with the retention time and resolution between NVP and internal standard carbamazepine as key responses. Chromatographic separation was achieved on a C₁₈ column using an isocratic mobile phase of water and acetonitrile (57.5:42.5 v/v), a 1.0 mL/min flow rate, and detection at 280 nm. The method was validated per ICH Q2 (R1) guidelines for all parameters including repeatability, intermediate precision, accuracy (recovery/bias), and robustness, in addition to specificity, linearity, LOD and LOQ.

Results and Discussion: The method demonstrated specificity, linearity (0.5–200 μg/mL), a detection limit of 0.033 μg/mL, and quantification limit of 0.5 μg/mL. The method was precise, accurate, and robust. Stress studies suggested stability-indicating performance under the tested conditions with no observed interference at the analyte retention time at 280 nm. Application to commercial and in-house formulations confirmed its suitability for routine analysis. This work highlights the value of AQbD in developing cost-effective, high-performing analytical methods for pharmaceutical analysis.

1 Introduction

Nevirapine (NVP) is an adipyrido-diazepinone compound, with an IUPAC name 11-cyclopropyl-4-methyl-5, 11-dihydro-6H-dipyrido (3, 2-b: 2′, 3′-e) (1, 4) diazepin-6-one (Sriram et al., 2006) and is classified as a non-nucleoside reverse transcriptase inhibitor (NNRTI) which is often used in tandem with other compounds for the suppression of HIV to halt development of AIDS (Lange, 2002). NVP was the first NNRTI approved by the Food and Drug Administration (FDA) for the treatment of HIV-1 infections and requires frequent dosing. NVP has a toxicity profile which includes serious side effects such as dermatological reactions, Stevens-Johnson Syndrome (SJS) and serious hepatotoxicity (Sulkowski et al., 2002). Presently, most NVP-based regimens require twice daily dosing and are associated with an inability to maintain drug levels at a sufficiently high concentration which, in part, has contributed to the occurrence of multi-drug resistant strains of HIV. To overcome some of these therapeutic challenges novel drug delivery systems have been proposed for the delivery of NVP. Our research group developed and optimised NVP-containing niosomes stabilised using dihexadecyl phosphate (DCP) and cholesterol in combination with Span® 20 or Span® 80 (Witika and Walker, 2019; 2021).

The analysis of nevirapine has been previously achieved using gradient elution (Mustafa et al., 2014), ion-pair RP-HPLC (Van Heeswijk et al., 1998), isocratic RP-HPLC (Pav et al., 1999; Hollanders et al., 2000; Kappelhoff et al., 2003; Ramachandran et al., 2006; Hamrapurkar et al., 2010; Kumar et al., 2010), gas chromatography-mass spectrophotometry (Vogel et al., 2010) and liquid chromatography-mass spectrophotometry (Rentsch, 2003; Sichilongo et al., 2014). Most of these methods have been used for the determination and quantitation of NVP in plasma (Pav et al., 1999; Hollanders et al., 2000; Kappelhoff et al., 2003), as bulk drug (Kappelhoff et al., 2003; Hamrapurkar et al., 2010; Ravisankar and Rao, 2013) and in dosage forms (Kappelhoff et al., 2003; Ravisankar and Rao, 2013) and require long analysis times which may not be suitable for routine laboratory use where a large number of samples may be required to be analysed within a short period.

Traditionally, the steps involved in method development entail a trial-and-error approach executed by evaluating the impact of one-factor-at-a-time (OFAT) on a separation. This approach generally requires a large set of experiments to be conducted and does not permit the evaluation of interactions between the factors evaluated, which may compromise the development and optimization process of a method (Bezerra et al., 2008; Fukuda et al., 2018). To circumvent the limitations of using an OFAT approach, a design of experiments (DoE) model, which is capable of generating results, including interaction effects, with fewer experimental runs (Bezerra et al., 2008) was used in this study.

The concept of analytical quality by design (AQbD) in analytical method development is a key aspectf in the area of pharmaceutical analysis. The AQbD approach places emphasis on science- and risk-based understanding of critical parameters which may affect method performance and results in the development of a robust method (Beg et al., 2012; 2015). The use of DoE is the cornerstone of pharmaceutical and analytical QbD (Bezerra et al., 2008).

A Central Composite Design (CCD) used for the optimization of response surface methodology (RSM) tools is among the most popular second-order designs as five (5) levels of each input factor are possible to evaluate using a reduced number of experiments when compared to three-level full factorial designs (Bezerra et al., 2008).

The present study aimed to develop and optimise a rapid, simple, selective, accurate and precise stability indicating RP-HPLC method for the analysis of NVP in bulk and tablet that is better than existing methods and capable of determining the NVP content in novel drug delivery platforms such as niosomes using CCD and RSM.

2 Materials and methods

2.1 Materials

NVP was donated by Aspen Pharmacare® (Port Elizabeth, South Africa). The methanol (MeOH) UV cut off 215 nm used to prepare stock solutions of NVP and internal standard and the HPLC grade acetonitrile 200 far UV Romil-SpS™ Super Purity Solvent (ACN) used to prepare mobile phase were purchased from Microsep® Ltd. (Port Elizabeth, South Africa). Carbamazepine (CBZ), which was used as the internal standard, was purchased from Sigma Aldrich® (St. Louis, United States of America). A Milli-Q Academic A10 water purification system, consisting of an Ion-X® ion-exchange cartridge and a quantum EX-ultrapore Organex® cartridge equipped with a 0.22 µm Millipak® 40 sterile filter (Burlington, MA, United States of America) was used to purify the HPLC grade water.

2.2 Methods

2.2.1 Instrumentation

The HPLC system utilised was a Waters® Alliance Model 2695 separation module, which included a solvent delivery module, auto-sampler, online degasser, and a model 2489 UV-vis detector (Milford, United States of America). The acquisition, processing, and reporting of data were conducted utilising Waters® Empower 3 software (Milford, United States of America). The stationary phase utilised was a Phenomenex Luna® C₁₈ (5 μm, 250 mm × 4.6 mm i. d) column (Separations, Randburg, South Africa). Detection was performed at 280 nm to provide a strong and stable UV response for nevirapine and the internal standard with an acceptable baseline under the selected chromatographic conditions. A Phenomenex Luna® C₁₈ (5 μm, 250 mm × 4.6 mm i. d.) column was selected as a robust reversed-phase stationary phase offering sufficient efficiency and resolution for separating nevirapine, carbamazepine, formulation/excipient components, and potential degradants, which is required for a stability-indicating assay.

2.2.2 Preparation of stock solutions

A standard stock solution was prepared by precisely weighing 200 mg of NVP with a Mettler® Model AE163 top-loading balance (Zurich, Switzerland) and quantitatively transferred to a 200 mL A-grade volumetric flask. The volumetric flask was subsequently filled to volume with MeOH. The solution underwent sonication for a duration of 10 min with a Branson® B12 sonicator (Shelton, United States of America), resulting in a concentration of 1 mg/mL. The standard stock solution facilitated the preparation of solutions within the concentration range of 0.5–200 μg/mL through a methodical serial dilution process utilising A-grade glassware.

2.2.3 Selection of internal standard

To reduce system and procedural variability that could occur during sample preparation, as a result of analytical procedure, or because of equipment variability, internal standards are utilised during quantitative analysis (Akpino, 1982). An internal standard enhances the precision of an analytical process by offsetting daily instrument fluctuations and inconsistent injection volumes (Lindholm et al., 2003).

Carbamazepine (CBZ) has commonly been used as an internal standard (Hamrapurkar et al., 2010; Mustafa et al., 2014) and oxcarbamezepine has also been used (Sarkar et al., 2006). Possible internal standards that were considered included efavirenz, tenofovir disoproxil fumarate and CBZ. CBZ resolved well and had a retention time of 7.12 min prior to method optimisation. Consequently, CBZ was chosen as the IS for these studies. The structures of NVP and CBZ are provided in Figure 1.

Figure 1

Figure 1. Chemical structures of NVP and CBZ.

2.2.4 Preparation of internal standard solution

A stock solution of internal standard was prepared by accurately weighing 200 mg carbamazepine (CBZ) using a Mettler® Model AE163 top-loading analytical balance (Zurich, Switzerland) and quantitatively transferred to a 200 mL A-grade volumetric flask. The volumetric flask was then made up to volume with MeOH and sonicated for 10 min using a Branson® B12 sonicator (Shelton, USA) to produce a solution of 1 mg/mL. Subsequently, 10 mL of the stock solution was transferred to a 100 mL A-grade volumetric flask and diluted with the mobile phase to achieve a concentration of approximately 100 μg/ml. A 0.75 mL aliquot of the 100 μg/mL CBZ was combined with 0.75 mL of the nevirapine (NVP) solution and stirred before analysis.

2.2.5 Preparation of mobile phase

The mobile phase used was comprised of acetonitrile and HPLC-grade water. The individual solvents were then transferred to a 1000 mL Scott® Duran bottle (Wertheim, Germany) and placed on separate lines of the HPLC system and mixed online for the analysis. The mobile phase was not recycled during analysis.

2.2.6 Experimental design

A CCD design was selected for the optimisation of the approach developed for NVP analysis. Initial studies suggested that three distinct input parameters, namely flow rate (X₁), organic solvent composition (X₂), and column temperature (X₃), were significant. The observed responses were the retention times of NVP (Y₁) and the resolution factor (Rs) (Y₂). The experiments conducted for the CCD are summarized in Table 1. To minimise any potential bias, all trials were performed in triplicate and randomised order.

Table 1

Table 1. Summary of CCD experiments performed, actual code and responses observed.

Factor coding was defined as A = flow rate, B = organic solvent (acetonitrile) content in the mobile phase, and C = column temperature. Model terms AB, AC and BC represent two-factor interactions, while A², B² and C² represent quadratic effects.

2.2.6.1 Method optimisation

Numerical optimisation was performed to identify method conditions that best satisfy the predefined analytical goals and constraints for the chromatographic performance responses. A desirability-based approach was implemented in Design-Expert® to explore the factor space and select an overall solution. The following factor constraints were applied with equal weights and importance set to 3: flow rate (A) targeted at 1.00 mL/min organic solvent fraction (B) constrained within 40%–60% (v/v), and column temperature (C) constrained within 25 °C–35 °C. Response goals were defined as minimising the retention time of nevirapine (RT NVP; 4.00–6.36 min) and maximising chromatographic resolution (Rs; 1.96–24.07) to support separation and method robustness. Although carbamazepine was used as an internal standard in the chromatographic method, its retention time (RT CBZ) was treated as a system suitability/control criterion with the upper limit set to 10 min rather than a primary optimisation response and is therefore not emphasised as a formal response variable. The recommended operating conditions were verified experimentally by running the method in replicate (n = 5) under the selected set-point.

2.2.7 Method validation

Method validation was performed in accordance with ICH guidance to confirm that the developed RP-HPLC procedure is suitable for its intended purpose in the quantitation of nevirapine in bulk material, tablets and niosome formulations. Validation parameters included precision, linearity and range, accuracy, limits of detection and quantitation, specificity and assay applicability (Causey et al., 1990; Green, 1996; Wood, 1999; Food and Drug Administration, 2015).

All validation experiments were conducted using the CCD-optimised chromatographic conditions. Unless stated otherwise, measurements were performed in triplicate (n = 3) and results are reported as mean values.

2.2.7.1 Precision

Precision describes the degree of agreement among individual results obtained when the method is applied repeatedly to multiple samples of a homogeneous matrix under prescribed conditions (Causey et al., 1990; Clarke, 1994). In this study, precision was assessed as repeatability (intra-day) and inter-day precision. Results were expressed as %RSD of NVP-to-IS peak area ratios. The acceptance criterion was set at ≤ 5 %RSD, which is appropriate for pharmaceutical dosage forms where biological-matrix variability is not applicable (Food and Drug Administration, 2015).

Repeatability (intra-day): Standard solutions across the calibration range were analysed on the same day in triplicate (n = 3), and %RSD of peak area ratios was calculated.

Inter-day precision: The same calibration standards were analysed in triplicate (n = 3) across three consecutive days, and %RSD values were determined to assess inter-day variability.

Reproducibility (between laboratories/operators) was not evaluated because the method was applied within a single laboratory by one analyst using the same instrument, and inter-day precision sufficiently addressed precision requirements for this context.

2.2.7.2 Linearity and range

Linearity was evaluated using least-squares regression of NVP concentration versus response (peak area ratio) over the specified working range. The coefficient of determination (R²) and regression equation were used to confirm proportionality across the calibration range (Causey et al., 1990; Food and Drug Administration, 2001). The range was defined as the interval between the lowest and highest concentrations that could be quantified with acceptable precision and accuracy.

2.2.7.3 Accuracy

Accuracy reflects the closeness of the measured value to the true value and was assessed using recovery experiments at three concentration levels (low, medium and high) spanning the calibration range (Causey et al., 1990; Green, 1996; Wood, 1999; Food and Drug Administration, 2001; Ermer and John, 2005). Samples were prepared at each level and analysed in triplicate (n = 3), with results reported as % recovery, % bias, and % RSD (Hokanson, 1994; Green, 1996).

2.2.7.4 Limits of detection and quantitation

The LOQ was defined as the lowest concentration that could be quantified with acceptable precision (≤5%RSD) and accuracy (Hokanson, 1994). The LOD was defined as the lowest concentration producing a measurable response but not meeting the requirements for accurate quantitation (Hokanson, 1994; Paino and Moore, 1999). LOQ was determined experimentally using the precision-based approach (≤5%RSD), and the LOD was calculated as 30% of the LOQ by convention (Paino and Moore, 1999).

2.2.7.5 Specificity

Specificity was assessed by demonstrating the absence of interference at the retention time of nevirapine and the internal standard in chromatograms of blank/mobile phase, reference standard solutions, and tablet sample solutions. Chromatograms of nevirapine standards were compared with chromatograms obtained from commercially available Aspen® nevirapine tablets to confirm that excipients did not interfere with the analyte response (Vessman, 1996; Rosing et al., 2000).

2.2.7.6 Assay and entrapment efficiency

2.2.7.6.1 Assay of commercial and in-house developed sustained release tablets

The suitability of the method for analysing NVP in dosage forms was confirmed by analyzing commercially available Aspen® NVP 200 mg tablets as well as in-house developed 400 mg sustained release tablets (Mwila, 2013) using the proposed technique. Twenty tablets were weighed and pulverised using a mortar and pestle. A quantity of powder equal to 100 mg was transferred into a 100 mL A-grade volumetric flask. Approximately 50 mL of methanol was added, and the mixture was subjected to sonication for 10 min before adjusting the volume with the mobile phase. The solution was then filtered with 0.45 µm HVLP Millipore® filter membrane and diluted with mobile phase to 100 μg/mL prior to analysis. The experiments were performed in triplicate.

2.2.7.6.2 Preparation of niosomes niosome samples for analysis

NVP niosomes were manufactured using a previously described thin layer hydration method (Azmin et al., 1985; Baillie et al., 2011) with slight modification and optimized using a non-rotatable Box-Behnken Design (BBD) to investigate the impact of four formulation and process variables on the Critical Quality Attributes (CQA) of NVP niosomes (Witika, 2017; Witika and Walker, 2019; 2021).

Briefly, a weight equivalent to 40 mg of NVP was accurately weighed and transferred to a round-bottomed flask and different molar ratios of surfactant, cholesterol and DCP were added. The powder was dissolved using 10 mL chloroform and methanol in a 90/10% v/v solution. The solvent was evaporated under vacuum using a Buchi™ R-215 rotary evaporator (Buchi™ Laboratories, Switzerland) for 45 min. Thereafter, the dried lipid layer was hydrated with 10 mL phosphate buffered saline (PBS) at pH 7.4. Hydration was conducted by rotating the sample at a temperature of 70 °C at a set speed for a specific period, as defined by the BBD. Following hydration, the suspension was annealed overnight at 4 °C (Witika, 2017; Witika and Walker, 2019; 2021).

Entrapment efficiency (EE) was determined by centrifuging 1 mL of each suspension using an Eppendorf 3154-C centrifuge (Hamburg, Germany) at 14,000 x g for 1 h. The supernatant was then decanted into a 10 mL A-grade volumetric flask and the pellet was washed using Milli-Q® water after which the sample was centrifuged at 14,000 g for a further 30 min to ensure all free NVP was removed. The supernatant was then decanted into the same A-grade volumetric flask, diluted with methanol and analysed. The entrapment efficiency was confirmed by destroying the niosome pellet at the base of the centrifuge tube with n-propanol and sonicating for 1 h using a Branson 8510 sonicator (Connecticut, USA). The solution was then decanted into a 100 mL A-grade volumetric flask and made up to volume with MeOH prior to analysis. Stability studies.

2.2.8 Forced degradation studies

2.2.8.1 Oxidative, acidic and alkali

Three separate 100 mL A-grade volumetric flasks were each filled with 100 mg samples of NVP. Forty millilitres of MeOH were added to each flask, and the solutions were subjected to sonication for 10 minutes. The solutions were prepared to a final volume using 30% v/v H₂O₂ (Allied Drug Company Ltd., Durban, South Africa), 0.1 M HCl, and 0.1 M NaOH for the oxidative, acidic, and alkaline degradation studies, respectively. The samples underwent reflux for 8 h at a temperature of 90 °C. Aliquots of 1.2 mL were collected at 2, 4, 6, and 8 h and diluted to a theoretical concentration of 50 μg/mL using mobile phase in 10 mL A-grade volumetric flasks. Aliquots of 0.75 mL were combined with 0.75 mL of the internal standard solution and vortexed before analysis via the RP-HPLC method outlined in this document. The experiments were conducted in triplicate.

2.2.8.2 Neutral hydrolytic studies

Neutral hydrolytic studies involved weighing 100 mg of NVP and transferring it into a 100 mL A-grade volumetric flask. Subsequently, 40 mL of MeOH was added, the solution was sonicated for 10 min, and the volume was adjusted with HPLC grade water. The sample was refluxed at 90 °C for 8 h, with 1.2 mL aliquots collected at 2, 4, 6, and 8 h, subsequently diluted to a concentration of 50 μg/mL using the mobile phase in a 10 mL A-grade volumetric flask. Aliquots of 0.75 mL were collected and combined with 0.75 mL of the IS solution, followed by vortexing before analysis using RP-HPLC.

2.2.8.3 Photodegradation studies

Approximately 100 mg NVP was accurately weighed and transferred into a 100 mL A-grade volumetric flask. Methanol (40 mL) was added and the solution was sonicated for 10 min and then made up to volume with mobile phase. The sample was exposed to light of 500 w/m2 at a temperature of 27 °C using a model CPS + SUNTEST® Weathering unit (Linsengericht, Germany) for 8 h. Sample aliquots of 1.2 mL were collected at 2, 4, 6 and 8 h and diluted to a concentration of 50 μg/mL with mobile phase in a 10 mL volumetric flask. Sample aliquots of 0.75 mL were then collected and 0.75 mL of internal standard solution added and the solutions vortexed prior to analysis by HPLC.

3 Results

3.1 Central Composite Design

Twenty experiments were performed as required for the CCD and a summary of the experiments and response factors are listed in Table 2. All experiments were conducted in a randomised fashion which resulted in simplified data sets being obtained.

Table 2

Table 2. CCD experiments and results for analysis of NVP.

The responses presented in Table 2 were assessed utilising version 8.0.2 of Design Expert® software (Minneapolis, MN, USA), and the data were analysed using various models. The effectiveness of each model in representing the relationship between the independent input variables and the monitored responses was assessed, with ANOVA employed to identify significant parameters.

3.1.1 Evaluation of model adequacy for retention time of NVP

Table 3 summarises the values of the factors considered to determine the model’s adequacy. The most critical metrics for determining model adequacy are the model F-value, coefficient of variance, acceptable precision, PRESS, and R² values.

Table 3

Table 3. Summary of model parameters and values used to evaluate adequacy of the model for resolution factor.

Table 4 summarises the results of the ANOVA analysis performed following the CCD trials, as well as the major factors influencing NVP retention. Values of “P > F” < 0.0500 suggest that the model terms are significant.

Table 4

Table 4. ANOVA for Response Surface Quadratic Model Analysis of variance table (Partial sum of squares - Type III) for the retention time of NVP.

3.1.2 Evaluation of model accuracy for R_s

Table 5 provides a summary of the findings used to determine R_s. Every parameter taken into account when evaluating the model’s suitability showed that the chosen quadratic model was suitable for navigating the design space.

Table 5

Table 5. Model parameters affecting resolution factor navigate the design space for this separation.

Table 6 presents the results of the ANOVA data analysis for the CCD experiments of the parameters that substantially affect the resolution between NVP and CBZ. The R_s for CBZ and NVP was shown to be influenced by the organic solvent concentration and flow rate, and model terms with a p-value <0.05 were deemed significant. The resolution factor was also significantly impacted by concurrent variations in the concentration of organic solvent and flow rate and the quadratic term B² was also significant, indicating curvature in the relationship between organic solvent composition and R_s across the explored range.

Table 6

Table 6. ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III] for the resolution factor.

3.1.3 Method optimisation

The optimised chromatographic conditions yielded a separation with retention times of NVP and CBZ at 4.10 ± 0.014 min and 6.7 ± 0.014 min, respectively, and a resolution factor of 13 ± 0.012 (n = 5). The optimal chromatographic conditions established for the quantification of NVP as well as the predicted and actual responses are presented in Table 7. These conditions were selected based on UV response at 280 nm and C₁₈ column performance to achieve adequate retention, peak shape and resolution. Figure 2 illustrates a conventional chromatogram depicting the separation of NVP and CBZ.

Table 7

Table 7. Optimised chromatographic conditions for the analysis of NVP and Predicted vs. Experimental values.

Figure 2

Figure 2. Typical chromatograms of NVP (200 μg/mL) and CBZ (100 μg/mL) using the optimized method.

3.1.4 Method validation

3.1.4.1 Linearity

A graph illustrating the peak area ratio of NVP/CBZ against the concentration of NVP produced a calibration curve with a slope of 0.0059, a y-intercept of 0.0066, and a correlation coefficient of 0.9994, as shown in Supplementary Figure S2. The results demonstrate that the approach exhibited linearity across the examined concentration range. The exceptional precision of the data rendered the standard deviation bars invisible on the plot.

3.1.4.2 Precision

3.1.4.2.1 Repeatability

The results for repeatability are summarized in Table 8. The % RSD for results is <5% which was the acceptable value for these experiments.

Table 8

Table 8. Results of repeatability studies.

3.1.4.2.2 Intermediate precision

The data obtained for intermediate precision are provided in Table 9. An RSD % <5% suggests that this approach is suitable for analysing NVP on different days.

Table 9

Table 9. Results of intermediate precision studies.

3.1.4.2.3 Accuracy

A summary of the results of accuracy studies is listed in Table 10 and indicates the method is accurate, with all % RSD and % Bias values <5%.

Table 10

Table 10. Results of accuracy studies.

3.1.4.3 LOQ and LOD

The LOQ was determined to be 0.5 μg/mL, with a corresponding % RSD of 0.65%. By convention, the LOD was set at 0.033 μg/mL.

3.1.4.4 Specificity

The specificity studies indicated that the NVP peak was not affected by the excipients present in both commercially available Aspen® nevirapine tablets and the in-house developed nevirapine tablets (Figure 3). Consequently, the method is considered precise for the examination of NVP in tablets and niosomal formulations.

Figure 3

Figure 3. Typical chromatogram following analysis of commercially available 200 mg Aspen^® nevirapine tablets (A) and in-house developed sustained release NVP tablets (Mwila, 2013) (B).

3.1.4.5 Assay and entrapment efficiency

3.1.4.5.1 Commercial and in-house sustained release tablet assay

The mean amount of NVP was found to be 98.35% ± 0.7435% of the label claim for the Aspen® NVP 200 mg tablets assessed while in the in-house developed sustained release tablet the label claims was found to be 99.13% ± 0.8325%.

The typical chromatogram of the tablet assay is provided in Figure 3.

3.1.4.5.2 Niosome entrapment efficiency

To demonstrate applicability of the proposed RP-HPLC method to vesicular matrices, the method was applied to quantify nevirapine (NVP) during determination of niosome entrapment efficiency (EE). Free (unentrapped) NVP was quantified in the supernatant, while entrapped NVP was quantified after disruption of the recovered pellet. Entrapment efficiency was calculated as:

$\%\text{EE}=\frac{\text{Entrapped}\text{NVP}}{\text{Total}\text{NVP}}\times 100$

Using this approach, the optimised NVP niosomes previously developed using Span-based systems achieved high %EE values (e.g., 96.8% and 98.0% for optimised Span® 20 and Span® 80 systems, respectively), confirming that the method is suitable for quantitation of NVP in niosome formulations (Witika and Walker, 2019).

In complementary excipient-screening work, %EE remained high even in the presence of cholesterol (e.g., 92.4%–94.1% at 50 µmol cholesterol, depending on surfactant), further supporting method applicability to niosomal matrices (Witika and Walker, 2021). A typical chromatogram of the quantitation of NVP from a niosome is depicted in Supplementary Figure S1.

3.1.5 Forced degradation studies

3.1.5.1 Oxidative degradation

NVP degraded by 15.0% ± 1.12% following the reflux for 8 h in 30% v/v H₂O₂. However, the chromatograms revealed no evidence of degradation peaks in all samples which agrees with previously reported results (Mwila, 2013).

3.1.5.2 Acidic degradation

NVP exhibited a degradation of 8.7% ± 0.87% in 0.1 M HCl following refluxing at 90 °C for 8 h, with no degradation peaks seen during the study period.

3.1.5.3 Alkali degradation

Following exposure to 0.1 M NaOH and refluxing at 90 °C for 8 h, degradation by approximately 11.9% ± 1.07% was observed with no degradation peaks observed for the duration of the study.

3.1.5.4 Neutral hydrolytic degradation

NVP demonstrated stability when subjected to reflux under neutral conditions at an elevated temperature of 90 °C.

3.1.5.5 Photodegradation

NVP demonstrated stability after being subjected to light conditions of 500 w/m² at 27 °C for a duration of 8 h.

The findings indicate a reduction in the % recovery of NVP following exposure to 30% v/v H₂O₂, 0.1 M HCl, and 0.1 M NaOH, along with refluxing at 90 °C for 8 h, signifying that deterioration had occurred. The % recovery of NVP remained mostly constant following exposure to neutral hydrolytic conditions at 90 °C and 500 w/m² at 27 °C for 8 h, indicating that NVP was stable under these conditions.

The outcomes of stability experiments regarding % recovery under various stress conditions at distinct time intervals are shown in Table 11.

Table 11

Table 11. Stability of NVP following stress testing.

4 Discussion

4.1 Evaluation of model accuracy for retention time of NVP

Regarding the ANOVA components (Table 4), the Residual sum of squares represents unexplained variability after fitting the model and was very small relative to the Cor Total variability, indicating that the fitted quadratic model explains the majority of the retention-time variation. The Lack of Fit term reflects any systematic deviation between the model and experimental data beyond replicate variability, whereas Pure Error represents replicate-to-replicate variability at identical factor settings. Due to the pure error being effectively zero in this dataset, due to very high repeatability at replicated points, the formal lack-of-fit partitioning becomes less informative. Overall model adequacy is therefore supported by the strong statistical diagnostics viz R² = 0.9934, Adj-R² = 0.9874, Pred-R² = 0.9497, low CV = 2.30%, and high Adeq Precision = 43.637 (Table 3).

The correlation between the independent input variables and the retention time of NVP is a function of the interaction of the stated parameters and is explained by Equation 1.

$RTNVP=4.08-0.63A-0.77B-0.014C+0.079AB+0.000AC+0.000BC+0.099A^2+0.28B^2-0.017C^2(1)$

The results generated review linear contributions of mobile phase organic composition and flow rate, as well as quadratic interactions thereof, had significant and antagonistic effects on the retention time of NVP as indicated by the negative sign of the model terms in the equation.

The flow rate, A, and organic solvent content, B, significantly influenced the retention time of NVP. Raising the organic solvent content leads to a decrease in retention time, as anticipated. This phenomenon can be explained by a diminished hydrophobic interaction between NVP and the stationary phase of the column (Wheeler et al., 1993; Snyder et al., 1997; Peiró-Vila et al., 2024). The increase in flow rate correspondingly decreased the retention time of NVP, a phenomenon that can be explained by the enhanced elution rate associated with a higher mobile phase flow rate.

The influence of column temperature on retention time was minimal. The equation indicates a negative sign on the parameter −0.014C, implying that an increase in temperature leads to decreased retention times. This phenomenon can be linked to a reduction in the viscosity of the mobile phase (Snyder et al., 1997).

The quadratic terms A² and B² indicate measurable curvature (non-linearity) in the response surface, meaning that the magnitude of the A and B effects is not strictly linear across the full investigated ranges which is expected in chromatographic DoE/retention modelling when factor ranges are wide enough for curvature to become detectable (den Uijl et al., 2021). The AB interaction term is small but non-zero, suggesting a modest dependence of the effect of organic solvent on RT NVP on the flow rate setting (and vice versa). In contrast, AC and BC are effectively zero in this model, indicating negligible interaction contributions involving temperature within the explored range.

4.1.1 Response surface model plots for retention time of NVP

The correlation among the key factors and the retention time of NVP can be illustrated through 3-D plots, as depicted in Figure 4. An increase in the concentration of organic solvent, while maintaining a constant flow rate, led to a decrease in retention time. The retention time of NVP decreases in a nearly linear fashion as the concentration of the organic solvent increases, assuming the column temperature remains constant. The 3-D plots demonstrate that the column temperature has a negligible impact on the retention time of NVP. An increase in both organic solvent concentration and column temperature results in a reduction of retention time.

Figure 4

Figure 4. 3D response surface plot depicting the impact of column temperature and flow rate (A) organic solvent content and flow rate (B) and column temperature and organic solvent content (C) on the retention time of NVP.

4.2 Evaluation of model accuracy for R_s

As with retention time, the near-zero pure error reflects high repeatability for replicated runs, limiting the interpretability of the lack-of-fit partitioning. The selected quadratic model is nevertheless strongly supported by its overall performance indicators: R² = 0.9963, Adj-R² = 0.9930, Pred-R² = 0.9723, CV = 5.03%, and Adeq Precision = 65.351 (Table 5), together with a highly significant model (p < 0.0001) (Table 6).

The fitted quadratic model describing the effect of the coded factors on the resolution response is given in Equation 2:

$R_s=8.9349-0.5969\mathrm{A}-6.6097\mathrm{B}+0.0091\mathrm{C}+0.2570\text{AB}-0.0008\text{AC}+0.0013\text{BC}-0.1391{\mathrm{A}}^2+1.2762{\mathrm{B}}^2-0.1889{\mathrm{C}}^2(2)$

Within the investigated design space, resolution was primarily governed by organic solvent content (B) and flow rate (A), while column temperature (C) exerted a negligible effect. Response surface analysis (Figure 5) confirmed that increasing the organic solvent fraction reduced R_s, and that this relationship was non-linear across the studied range, consistent with the significant B² term. Increasing flow rate likewise decreased R_s, reflecting reduced time for separation under faster elution conditions. Interactions involving temperature (AC and BC) were minimal, in agreement with their non-significant contributions in the ANOVA. Overall, the model and response surfaces highlight organic solvent composition as the main lever for maintaining adequate resolution, with flow rate acting as a secondary but significant contributor.

Figure 5

Figure 5. 3D response surface plot depicting the impact of column temperature and flow rate (A) organic solvent content and flow rate (B) and column temperature and organic solvent content (C) on the resolution factor.

4.3 Comparative evaluation with published HPLC methods for nevirapine

Several RP-HPLC methods for the determination of nevirapine in bulk drug and dosage forms have previously been reported (Table 12). The majority of reported RP-HPLC techniques for nevirapine predominantly utilise buffer-based mobile phases (phosphate or acetate) and, in certain instances, gradient elution systems. Although successful, these methods are operationally intricate: buffer preparation is laborious, may reduce column longevity due to salt precipitation, and produces excess laboratory waste. Conversely, the current technique employs a straightforward isocratic system comprising solely acetonitrile and water, so mitigating these disadvantages and improving both sustainability and cost-efficiency.

Table 12

Table 12. Summary of existing HPLC methods NVP that analyse Tablet or Bulk.

Equally significant, the majority of previously documented approaches were formulated through trial-and-error optimisation and failed to integrate systematic Design of Experiments (DoE) or Analytical Quality by Design (AQbD) concepts. The lack of a specified design space constrains their reliability when utilised beyond restricted laboratory environments. In contrast, the current investigation utilised Central Composite Design (CCD) and Response Surface Methodology (RSM) to delineate factor interactions and guarantee consistent performance amidst minor fluctuations in flow rate, temperature, and solvent ratio.

With regard to elution efficiency, gradient methods often suffer from long run times and equilibration periods (≥14 min in (Kapoor et al., 2006; Samee et al., 2007)). Even isocratic buffer-based systems typically report retention times >7 min (Chan Li et al., 2000; Navaneethan et al., 2012). The present method achieves a shorter run time of <8 min, allowing high sample throughput in quality control environments. Furthermore, unlike most earlier methods, an internal standard (carbamazepine) was utilized here, significantly improving reproducibility and accuracy.

Analytical performance comparisons also favour the current method. While some reports demonstrated sensitivity at low µg/mL levels (e.g. LOQ = 0.24 μg/mL (Ravisankar and Rao, 2013)), most published methods had narrow linearity ranges (e.g. 2–10 μg/mL, 120–360 μg/mL). The present method covers a broad range of 0.5–200 μg/mL with excellent linearity (R² = 0.9994) and relatively low LOQ (0.5 μg/mL), making it versatile for both low-dose and high-concentration analyses.

Finally, while stability-indicating capability has been reported in selected studies these were primarily impurity-focused and not combined with AQbD principles. This method provides a distinctive blend of simplicity, robustness, sensitivity, and stability-indicating performance, and is further distinguished by its applicability to both commercial dosage forms and advanced drug delivery systems (niosomes), which are infrequently discussed in prior literature. A summary of how prior studies compare to this method is provided in Table 12.

5 Conclusion

The application of RSM for method optimization to determine the ideal chromatographic conditions for separating NVP with the IS, CBZ, facilitated the development of a method utilising a straightforward mobile phase without buffer, resulting in a more cost-effective approach compared to previously documented methods (Pav et al., 1999; Chan Li et al., 2000; Lopez et al., 2001; Anbazhagan et al., 2005; Kapoor et al., 2006; Shah et al., 2012; Ravisankar and Rao, 2013).

The absence of interfering peaks during the analysis of commercially available Aspen® NVP tablets as well as the in-house developed sustained release tablets is an indication that the method is specific for the analysis of NVP analysis in these tablets and can be used for niosomal formulations. The LOQ and LOD values obtained were very low indicating the sensitivity of the method developed over the existing HPLC methods (Pav et al., 1999; Chan Li et al., 2000; Lopez et al., 2001; Anbazhagan et al., 2005; Kapoor et al., 2006; Shah et al., 2012) for the analysis of NVP. The low values for % RSD of <5.0% for the accuracy and intra and inter-day precision studies reflect the adequacy of the analytical method.

The application of the concepts of AQbD to method development and validation is efficient and has universal application for the development of simple, reproducible, selective and rapid analytical methods.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions

BW: Visualization, Formal Analysis, Data curation, Writing – original draft, Software, Investigation. RW: Resources, Writing – original draft, Supervision.

Funding

The author(s) declared that financial support was received for this work and/or its publication. The authors express their gratitude to the Rhodes University Research Committee (RBW) and the Henderson Scholarship (BAW) for providing financial support.

Acknowledgements

The authors recognise the contribution of Aspen Pharmacare® Ltd. for providing a donation of NVP.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author BW declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/frans.2026.1735125/full#supplementary-material

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Glossary

A Flow rate (coded DoE factor)

A² Quadratic term for factor A

AB Interaction term between factors A and B

AC Interaction term between factors A and C

ACN Acetonitrile

Adj R² Adjusted coefficient of determination

AIDS Acquired immunodeficiency syndrome

ANOVA Analysis of variance

AQbD Analytical Quality by Design

B Organic solvent content (coded DoE factor; acetonitrile fraction)

B² Quadratic term for factor B

BBD Box–Behnken design

BC Interaction term between factors B and C

C Column temperature (coded DoE factor)

C² Quadratic term for factor C

C₁₈ Octadecylsilane (C₁₈) stationary phase

CBZ Carbamazepine

CCD Central composite design

CQA Critical quality attribute(s)

DCP Dicetyl phosphate

df Degrees of freedom

DoE Design of experiments

EE Entrapment efficiency

FDA U.S. Food and Drug Administration

HIV Human immunodeficiency virus

HPLC High-performance liquid chromatography

ICH International Council for Harmonisation

IS Internal standard

LOD Limit of detection

LOQ Limit of quantitation

MeOH Methanol

MODR Method operable design region

NNRTI Non-nucleoside reverse transcriptase inhibitor

NVP Nevirapine

OFAT One-factor-at-a-time

PBS Phosphate-buffered saline

Pred R² Predicted coefficient of determination

QbD Quality by Design

R² Coefficient of determination

RSD Relative standard deviation

%RSD Percent relative standard deviation

RP-HPLC Reversed-phase high-performance liquid chromatography

R_s Resolution

RT Retention time

RSM Response surface methodology

SD Standard deviation

SJS Stevens-Johnson syndrome

UV Ultraviolet

v/v Volume/volume