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INTRODUCTION
Diabetes mellitus is a long-term metabolic condition characterised by sustained increases in blood glucose concentration. As reported by the World Health Organization, around 830 million individuals globally are affected by diabetes mellitus.1 A recent study conducted by the Indian Council of Medical Research highlighted a high prevalence of both diabetes and prediabetes in India, estimating that nearly 101 million individuals are living with diabetes, while about 136 million are in the prediabetic stage. In addition, data from International Diabetes Federation indicate that India ranks second globally in terms of the number of people affected by type 2 diabetes mellitus, thereby contributing substantially to the global disease burden.2 IUPAC name of LIN is 8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-ynyl-3-methyl-1-[(4-methyl quinazolin-2-yl) methyl] purine-2,6-dione3 shown in Figure 1. LIN, a xanthine-based dipeptidyl peptidase-4 (DPP-4) inhibitor, is given orally once daily. It suppresses the enzymatic activity of DPP-4, leading to increased circulating levels of active incretin hormones, including glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide following food intake. This mechanism contributes to improved glycaemic control by enhancing insulin secretion from pancreatic β-cells while simultaneously reducing glucagon release from α-cells.4 Chemical name of DAP is (2S,3R,4R,5S,6R) -2-[4-chloro-3-[(4 ethoxy phenyl) methyl] phenyl]-6- hydroxy methyl) oxane-3,4,5-triol; (2S)- propane-1,2-diol; hydrate5 shown in Figure 1. DAP, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, is extensively utilised in the treatment of type 2 diabetes mellitus, providing effective glycaemic regulation along with additional cardiovascular and renal protective benefits.4 Prashant Kumthekar et al., in their study suggested that the fixed dose combination of and LIN 5 mg and DAP 10 mg tablet is superior to LIN 5 mg alone in terms of efficacy. The combination therapy represents a promising and effective approach for managing type 2 diabetes, resulting in notable improvements in glycaemic control. Additionally, both agents were generally well tolerated, with a low frequency of adverse events. This combination has gained significant therapeutic importance in the management of type 2 diabetes mellitus. Consequently, the development of reliable and sensitive analytical methods for their simultaneous quantification in bulk and pharmaceutical dosage forms has attracted considerable research interest.
A comprehensive literature review revealed that several UV spectrophotometric methods.6,11 have been widely investigated for quantifying LIN and DAP, both as individual analytes and in combination with other pharmaceutical agents. In addition, several HPLC methods have been developed for the estimation of DAP and its combinations with other drugs.12,14,16,23 , as well as for LIN and its combinations.24,32 Furthermore, one HPTLC method.15,33 has been reported. However, only a limited number of HPLC methods are available for the simultaneous determination of LIN and DAP.34,36
Existing methods predominantly rely on conventional trial-and-error approaches, lack QbD-based optimisation, do not include quantitative greenness assessment and solution stability studies. All reported methods for the estimation of LIN and DAP employ acetonitrile as the solvent, which is relatively expensive and consequently increases the overall cost of the method. Hence, there exists a clear need to develop a systematic, statistically optimised, cost-effective and efficient reversed phase high performance liquid chromatographic method with enhanced performance for the simultaneous quantification of LIN and DAP in pure drug substance and tablet dosage form.
MATERIALS AND METHODS
Chemicals and Reagents
Chromatography grade water, methanol and potassium dihydrogen phosphate were provided by Merck Life Sciences Pvt. Ltd., Mumbai, India. Nylon membrane filter (0.45 µm size) was made available from Merck Life Sciences Pvt. Ltd., Bangalore, India. Other chemicals utilised for this research work were analytical grade and solvents of HPLC grade.
Active pharmaceutical ingredient of LIN and DAP were obtained from Swapnroop Drugs and Pharmaceuticals, Chhatrapati Sambhajinagar, India. Tablet Juslina D -5/10 containing 5 mg of LIN and 10 mg of DAP, (Manufactured by Exemed Pharmaceuticals, GIDC, Vapi. Dist.- Valsad, Gujarat, India.) were bought from local pharmacy.
Instruments and software
Analysis of drug combination was performed by using HPLC System (HPLC 3000 series, Analytical Technologies Pvt. Ltd.,) with UV visible detector (UV - 3000M), HPLC workstation software was used for analytical data integration and utilised Cosmosil C18 column with dimensions 250 mm x 4.6 mm, 5 µm. UV visible Spectrophotometer (Analytical Technologies Ltd., 2012), sonicator (Labman Scientific Instruments, LMUC Series), analytical weighing balance (Wensar, DAB220), pH meter (Hanna Instruments, HI2211). Design expert was used for evaluation of experimental design study (Stat-Ease Inc., Minneapolis, USA, Version 13.0).
Selection of mobile phase
LIN and DAP were soluble in methanol. Potassium dihydrogen phosphate buffer at pH 2.8 was used to control the ionisation of LIN and DAP, improve peak symmetry, minimise silanol interactions, and achieve adequate resolution with methanol as organic modifier in RP-HPLC. Mobile phase comprising of methanol: Phosphate buffer (10 mM KH2PO4) pH 2.8 in the ratio 70:30%v/v was selected for quantitation of LIN and DAP since it gave sharp, well resolved peaks. Then eluting solvents was vacuum degassed and filtered by using 0.45µ size membrane filter.
Preparation of phosphate buffer
Weighed accurately 0.137 g of Potassium dihydrogen phosphate (KH2PO4) and dissolved in 100 mL of water. The pH was adjusted to 2.8 using dilute orthophosphoric acid and measured using a calibrated pH meter. Before analysis, the pH meter was standardised with certified pH 4.0 and 7.0 buffer solutions in accordance with the manufacturer’s instructions. The prepared buffer was subsequently passed through a 0.45 μm membrane filter and subjected to sonication before being used in the analysis. This solution was used in the mobile phase composition.
Preparation of standard stock solution
Weighed accurately 10 mg of LIN and DAP separately and dissolved in 10 mL of solvent (solvent was used as mobile phase) to produce 1000 ppm solution.
Preparation of sample solution
Accurately weighed twenty tablets and triturated utilising mortar and pestle. Weight of tablets was found to be 9.0 g and average weight of tablet was 204 mg. Powder analogous to 5 mg of LIN and 10 mg of DAP was accurately weighed and transferred in to 100 mL volumetric flask, dissolved in approximately 70 mL methanol, sonicated for 15 min, and filtered. An appropriate aliquot of the filtrate was further diluted with mobile phase (Methanol: Phosphate Buffer, 70:30 v/v) to obtain the required concentration for analysis.
Selection of wavelength for analysis
The overlain UV spectra of LIN and DAP were recorded in methanol over the wavelength range of 200-400 nm. Freshly prepared 10 µg/mL standard solutions of were analysed in U.V. visible spectrophotometer. LIN exhibited λmax at 224 nm, whereas DAP showed a characteristic λmax at 295 nm. The spectra intersected at 227 nm, indicating an isosbestic point where both analytes exhibited identical absorptivity. Therefore, 227 nm was selected as the analytical wavelength for simultaneous estimation.
HPLC method optimisation by AQbD approach
Analytical Target Profile (ATP)
Defining ATP is the most important step and first step while optimising analytical method by using experimental design technique. An ATP consists of performance characteristics for quality attributes including appropriate details on the product and process parameters to be determined with associated performance criteria.
Method Performance Characteristics (MPCs)
These are the method parameters that directly influence the ATP. Retention time, no. of theoretical plates used as method performance characteristics. MPCs should be controlled to obtain the desired output.
Analytical Procedure Parameter (APP)
These are factors or independent variables which having potential effect or they are critical for performance of method. Factors such as flow rate, mobile phase composition, pH are regarded as analytical procedure parameter.
Risk assessment
Risk assessment study was done by Ishikawa fishbone diagram and on the basis of literature available. Effect of critical procedure parameter on the dependent variable was evaluated. Effect of APP on MPC was studied in risk assessment.
Selection of Experimental Design
Box-Behnken Design (BBD) was utilised for method optimisation. It requires less experimental runs than Central Composite Designs (CCD) for the same number of independent variables, particularly when applying quadratic relationships. Hence BBD is less costly as compared to CCD. Therefore, BBD was implemented for optimisation HPLC technique. During making BBD three factors, two levels and five centre points were taken for study; 17 experimental runs were produced by design expert software which were performed (Table 1). Standard and sample preparations were made and subsequently injected into the chromatographic system. For both drugs Retention time, theoretical plates were measured as responses. The Design Expert software was utilised for evaluating the design of experiment study. (Stat-Ease Inc. Minneapolis, USA, Version 13.0). ANOVA with a quadratic approach was utilised to evaluate the coefficients and nature of the robustness. The significance of the independent variables was estimated by statistical technique ANOVA. Evaluation of the quadratic model’s significance was carried out based on the p-value, F-value, and goodness-of-fit statistics.
The relationship between APP and MPCs was described using a second-order polynomial equation generated by the Box-Behnken Design:
Where Y represents the response (e.g., retention time, number of theoretical plates), β₀ is the intercept, β₁-β₃ are the linear coefficients, β₁₂-β₂₃ are the interaction coefficients, β₁₁-β₃₃ are the quadratic coefficients, and A, B, and C represent the selected APP.
The significance of the model and individual coefficients was evaluated using ANOVA, and the optimised chromatographic conditions were obtained using the desirability function approach.
Method Operable Design Region (MODR)
The Method Operable Design Region (MODR) represents a dynamic, multidimensional domain in which the combined influence of independent variables maintains minimal variability in performance. It is defined using numerical optimisation techniques, with clearly established boundaries for each individual response to ensure consistent and reliable outcomes.37
Analytical Method Control Strategy
A structured control strategy, developed from in-depth method knowledge and development studies, that ensures the analytical procedure consistently delivers accurate, reliable, and robust performance across its entire lifecycle.
Solution Stability Study
Standard solution stability was evaluated by conducting dedicated stability studies. Triplicate standard solutions were kept at ambient temperature and analysed after 24 h (day 1) and 48 h (day 2), followed by calculation of %RSD to assess consistency. Mobile phase stability was examined by monitoring any changes in appearance, along with system suitability and repeatability, after storage on the benchtop for the same time intervals.38
Validation of Analytical Procedure
The optimised high-performance liquid chromatographic method was validated in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2 (R2) guidelines. Key performance characteristics evaluated included linearity, precision, accuracy, robustness, as well as the limits of detection and quantification.39
Assessment of Analytical Greenness
Two different ecological metrics were employed to assess the effect of developed technique on environment. Greenness AGREE, Complex GAPI and Analytical Eco-Scale was utilised to estimate the environmental compatibility. Evaluation is carried out by considering use of solvents, wastage produced and energy used during analytical procedure development.40,41
| Factors | Responses | ||||||
|---|---|---|---|---|---|---|---|
| Run | % Methanol | Flowrate | pH | RT of LIN | RT of DAP | TP of | TP of |
| 1 | 70 | 0.9 | 3 | 3.572 | 7.842 | 5546 | 11460 |
| 2 | 80 | 0.8 | 3 | 3.345 | 5.955 | 4116 | 11307 |
| 3 | 80 | 0.9 | 2.8 | 2.982 | 5.429 | 3544 | 10438 |
| 4 | 70 | 0.9 | 3 | 3.569 | 7.835 | 5551 | 11455 |
| 5 | 80 | 0.9 | 3.2 | 2.982 | 5.48 | 4635 | 10345 |
| 6 | 70 | 0.9 | 3 | 3.577 | 7.840 | 5539 | 11467 |
| 7 | 70 | 1 | 2.8 | 3.203 | 7.113 | 1883 | 11030 |
| 8 | 80 | 1 | 3 | 2.704 | 4.953 | 2029 | 9852 |
| 9 | 60 | 0.9 | 2.8 | 4.043 | 16.652 | 4345 | 12733 |
| 10 | 60 | 0.8 | 3 | 4.585 | 18.994 | 4658 | 13351 |
| 11 | 70 | 0.9 | 3 | 3.570 | 7.847 | 5545 | 11453 |
| 12 | 70 | 0.8 | 3.2 | 3.733 | 11.203 | 4733 | 12068 |
| 13 | 70 | 0.9 | 3 | 3.575 | 7.841 | 5549 | 11461 |
| 14 | 70 | 0.8 | 2.8 | 3.729 | 11.137 | 4309 | 12430 |
| 15 | 60 | 0.9 | 3.2 | 4.058 | 16.903 | 4514 | 13518 |
| 16 | 70 | 1 | 3.2 | 3.219 | 7.155 | 2160 | 10598 |
| 17 | 60 | 1 | 3 | 3.923 | 11.783 | 5488 | 12806 |
RESULTS
Analytical Target Profile (ATP)
The Analytical Target Profile of the proposed RP-HPLC method was to develop a simple, rapid, accurate, precise, robust analytical procedure for the simultaneous quantification of LIN and DAP in tablet dosage forms. The method was designed to achieve assay results within 98-102% of the label claim, precision with %RSD not exceeding 2%, adequate chromatographic resolution (resolution > 2), acceptable peak symmetry (tailing factor < 2), and a total chromatographic run time not exceeding 15 min.
Method Performance Characteristics (MPCs)
Retention time, theoretical plates were identified as method performance characteristics which were highly important parameters for method development.
Analytical Procedure Parameter (APP)
Factors or independent variables such as mobile phase composition (% organic phase i. e. methanol), flow rate and pH were recognised as analytical procedure parameter.
Risk assessment
Effect of analytical procedure parameter on the dependent variable was evaluated by using Ishikawa fishbone diagram and on the basis of literature available. Effect of APP on CQA was studied in risk assessment.
Experiments were performed as per Box Behnken Design using reverse phase HPLC. Retention time, theoretical plate, peak area, resolution etc. were assessed. Three independent variables selected found critical for analysis of LIN and DAP. The proposed design was statistically evaluated by ANOVA. The analysis provides contour plot, 3D response surface graph by plotting the response against other two factors while the third factor was kept maintained at a desired level, and representation of a response as two dimensional and three-dimensional response surface plots were shown in Figure 2. The p-value and F statistics values were noted for each dependent variable, confirming robustness of developed method at extended variations in conditions.
Retention time of optimised reversed phase liquid chromatographic method of LIN and DAP was 3.72 min, 11.13 min correspondingly. Resolution among two peaks was 23.27 confirming good separation. The corresponding peak areas were 309428 and 158064. Theoretical plates were measured to be 4309 and 12430, indicate column performance was efficient for the given conditions.
Optimised Chromatographic Condition for analysis
Optimised mobile phase by Box Behnken design consisting of methanol and phosphate buffer pH 2.8 in ratio 70:30 %v/v and flow rate was 0.8 mL/min. Cosmosil C18 column (250 mm x 4.6 mm; 5 µm) was utilised for chromatographic estimation. Detection wavelength was 227 nm which is isosbestic point. Run time was 15 min. Sample volume 20 µL was used. Run time 15 min. The different interaction and quadratic effects of the composition of mobile phase and flow rate on retention time, number of theoretical plates were analysed using ANOVA tool (Figure 3).
Design Space
MODR was established in the mobile phase composition 62 to 71% methanol, buffer pH 2.8 to 3.2 and flow rate is 0.8 to 1 mL/min consistently met all predefined MPC acceptance criteria. The best conditions were determined through numerical optimisation, yielding the solution with the maximum desirability value (Table 2).
Analytical Control Strategy
Percentage composition of organic phase i.e. percentage of methanol and flow rate were identified as critical points which needs to be controlled to get within limit MPC results.
Validation of Analytical Procedure
System Suitability
Prior to sample analysis, the chromatographic system was evaluated through system suitability testing to ensure that all instrumental and separation parameters were operating satisfactorily. Six consecutive injections of the standard solution were carried out, and the %RSD of the peak areas remained within acceptable criteria, demonstrating consistent repeatability. Column performance was verified by satisfactory theoretical plate values, while peak symmetry was indicated by appropriate tailing factors. In addition, adequate resolution between closely eluting peaks confirmed effective separation. Overall, these findings establish that the system is fit for routine analytical use (Table 3). The system suitability results demonstrated acceptable chromatographic performance for both analytes. LIN exhibited a tailing factor of 1.36, while DAP showed a tailing factor within the acceptable range. Although LIN is a basic drug and may exhibit secondary interactions with residual silanol groups of the stationary phase, the optimised mobile phase composition and pH provided satisfactory peak symmetry. The observed tailing factor was well below the acceptance limit of 2.0, indicating that the method is suitable for routine quantitative analysis.
Specificity
The specificity of the method was evaluated by examining blank, standard, and sample preparations. No interfering signals were detected at the retention times corresponding to the analytes, demonstrating that excipients and other matrix components did not influence the measurement. These findings confirm that the method is selective and suitable for the intended analysis, shown in Figure 4. The specificity of the proposed RP-HPLC method was demonstrated by comparing blank, standard, and sample chromatograms. The retention times of LIN and DAP in the sample solution were consistent with those observed in the standard solution. Minor additional peak observed in the sample chromatogram were attributed to formulation excipients and matrix constituents. These peaks were well resolved from the analyte peaks and did not interfere with the determination of either drug, confirming the specificity of the method.
Linearity
Method linearity was assessed using multiple standard solutions prepared at different concentration levels. For each analyte, a calibration plot was generated by correlating the measured chromatographic peak response with the corresponding concentration. The developed method exhibited linearity over the concentration varying from 10-50 μg/mL for LIN with R2 value 0.9994 and 20-100 μg/mL for DAP with R2 value 0.9996, indicating excellent proportionality between response and concentration (Figure 5).
Accuracy
The accuracy of the developed analytical procedure was determined using a standard-addition approach at three levels, namely 50%, 100%, and 150% of the nominal test concentration. Appropriate quantities of LIN and DAP reference standards were added to the previously analysed sample matrix, and each spiking level was examined in three independent determinations. The percentage recovery, standard deviation, and %RSD were calculated. The recoveries were found to be within acceptable limits, indicating that the method is accurate and free from interference of excipients. Low %RSD (>2%) values further confirmed the reliability of the method.
Precision
Precision of the method was assessed in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). For repeatability, multiple injections of the same concentration were analysed within a single day, while intermediate precision was evaluated on different days. Inter-day precision was assessed by analysing three independently prepared sample solutions at the working concentration on three consecutive days. The analyses were carried out under identical chromatographic conditions using the same instrument, column, mobile phase composition, flow rate, injection volume, and detection wavelength. The assay results obtained on different days were compared, and the percentage Relative Standard Deviation (%RSD) was calculated to evaluate method reproducibility. The %RSD values for peak area were found to be within acceptable limits, demonstrating that the method is precise and reproducible (Table 4).
Robustness
Robustness of the method was determined by making small deliberate variations in pH and detection wavelength. The effect of these changes on system suitability parameters and assay results was evaluated. The method remained unaffected by these minor variations, indicating its robustness and reliability during routine usage. The robustness of the optimised RP-HPLC procedure was assessed by intentionally modifying selected critical chromatographic conditions to determine the method’s ability to maintain consistent analytical performance. The parameters studied included mobile phase pH (±0.2 units), flow rate (±0.1 mL/min), methanol composition (±2%), and detection wavelength (±2 nm). Sample solutions were analysed under each modified condition, and the assay results along with system suitability parameters were recorded. The method was considered robust when the %RSD of assay results was less than 2.0% and no significant changes in retention time, peak symmetry, or theoretical plate count were observed (Table 4).
Limit of Detection and Limit of Quantification
The Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined according to ICH Q2(R2) guidelines using the standard deviation of the response (σ) and the slope of the calibration curve (S). The following equations were employed:
Where σ is the standard deviation of the intercepts of the regression lines and S is the slope of the corresponding calibration curve.
Assay
The assay of LIN and DAP in tablet dosage form was calculated using the external standard method by comparing the peak areas obtained from sample and standard solutions after applying the appropriate dilution and purity correction factors.
The percentage assay was calculated using the equation provided below.
Where:
AT = Peak area of test solution,
AS = Peak area of standard solutions,
WS = Weight of working standard (mg),
WT = Weight of sample taken (mg),
DS = Standard dilution factor,
DT = Sample dilution factor,
P = Purity of working standard (%),
Label Claim = Amount of drug claimed per tablet.
The proposed technique was adopted for the assay of tablet formulation. Analysis was done by injecting the blank, standard and sample in to the chromatographic system and peak areas of LIN and DAP were used for calculating the % assay by using the formula. The % purity of tablets of LIN and DAP was ascertained to be 99.63% and 100.13% respectively.
Result of solution stability
The mean peak areas obtained at Initial, 24 h, and 48 h were compared to evaluate solution stability. The overall %RSD was found to be 0.618%, and 0.0354 for LIN and DAP respectively, which is well below the acceptance criterion of 2.0%, indicating that the analytical solution remained stable for at least 48 h under the studied conditions (Table 5).
Assessment of Analytical Greenness
Greenness AGREE assessment
The developed HPLC procedure was scored 0.61 by using Greenness AGREE software indicates that the developed method is moderate green. AGREE scores range from 0 to 1. The GAPI pictogram revealed predominantly green and yellow zones, indicating acceptable environmental friendliness of the proposed HPLC method, with red zones mainly associated with the use of organic solvents. Greenness results were visually depicted in Figure 6. The environmental sustainability of the developed RP-HPLC procedure was evaluated using the AGREE (Analytical GREEnness Metric for analytical methods) assessment tool. The AGREE score ranges from 0 to 1, with higher values indicating better compliance with the twelve principles of Green Analytical Chemistry. In general, scores between 0.8 and 1.0 represent excellent greenness, scores between 0.6 and 0.8 indicate acceptable to good greenness, and scores below 0.6 suggest limited greenness. The obtained AGREE score of 0.61 demonstrates that the proposed method possesses an acceptable degree of environmental sustainability while maintaining suitable analytical performance for routine quality control applications.
Complex GAPI assessment
The Complex GAPI assessment revealed a predominance of green and yellow zones, indicating that most analytical steps exert low to moderate environmental impact. The red zones were mainly attributed to the use of methanol in the mobile phase and the associated solvent waste generated during chromatographic analysis. The proposed method offers environmental advantages compared with several previously reported methods that utilise acetonitrile as the principal organic component of the mobile phase. Acetonitrile is generally associated with higher toxicity, greater environmental burden, and increased disposal concerns compared with methanol. The replacement of acetonitrile with methanol, together with a relatively short analysis time and moderate solvent consumption, contributed to an improved environmental profile. Therefore, despite the presence of some red zones in the Complex GAPI pictogram, the overall assessment supports the acceptable greenness and sustainability of the developed method.
Analytical Eco-Scale assessment
The developed RP-HPLC procedure was additionally assessed for its environmental sustainability using the Analytical Eco-Scale (AES) approach. The Eco-Scale score was calculated by assigning penalty points for reagent hazards, solvent consumption, energy requirements, occupational hazards, and waste generation. The proposed method employed methanol-phosphate buffer (70:30, v/v) as the mobile phase, generated approximately 25 mL of waste per analysis, and required conventional HPLC instrumentation. The total penalty points were estimated to be 23, resulting in an Analytical Eco-Scale score of 77. According to the Eco-Scale classification, methods with scores greater than 75 are considered excellent green analytical procedures. Therefore, the developed RP-HPLC method can be classified as an environmentally sustainable analytical method, supporting the conclusions obtained from the AGREE and Complex GAPI assessments (Table 6).
| Dependent Variables | ANOVA | Fit statistics | |||
|---|---|---|---|---|---|
| p-value | F- value | Predicted R2 | Adjusted R2 | Adequate Precision | |
| Retention time (LIN) | 0.0001 | 122.04 | 0.8987 | 0.9855 | 40.70 |
| Retention time (DAP) | 0.0001 | 237.71 | 0.9478 | 0.9925 | 51.45 |
| Theoretical plate (LIN) | 0.0171 | 5.55 | 0.9670 | 0.7190 | 6.91 |
| Theoretical plate (DAP) | 0.0001 | 62.32 | 0.8840 | 0.9388 | 27.03 |
| Parameters | Results | Acceptance criteria | |
|---|---|---|---|
| LIN | DAP | ||
| Retention time (min) | 3.71 | 11.14 | NA |
| Tailing factor | 1.36 | 0.95 | NMT 2 |
| Theoretical plate counts | 6963 | 11602 | NLT 2000 |
| Resolution between LIN and DAP peak | - | 24.70 | NLT 2 |
| Percentage RSD for peak area (six replicate injections) | 0.1604 | 0.2510 | NMT 2% |
| Validation Parameters | LIN | DAP |
|---|---|---|
| Specificity | No interference | No interference |
| Linearity (correlation coefficient) | 0.9994 | 0.9996 |
| Range | 10-50µg/mL | 20-100µg/mL |
| Accuracy (%Recovery) | 0.1623 | 0.0719 |
| Level 1 (50%) | 100.2741 | 100.8620 |
| Level 2 (100%) | 100.1485 | 99.6728 |
| Level 3 (150%) | 100.0672 | 99.6722 |
| Precision - Intraday (%RSD) | 0.2761 | 0.0571 |
| Precision - Interday (%RSD) | 0.2944 | 0.0405 |
| LOD | 0.4624 | 0.2807 |
| LOQ | 1.401 | 0.8506 |
| Robustness - change in pH (%RSD) | 0.4947 | 0.3951 |
| Robustness - change in wavelength (%RSD) | 0.2173 | 0.7278 |
| LIN | Time | Conc. (µg/mL) | Area (Units) | Mean | SD | % SD | % RSD |
|---|---|---|---|---|---|---|---|
| Initial | 50 | 1965343 | 1977281 | 10410 | 0.5265 | 0.618 | |
| 50 | 1982029 | ||||||
| 50 | 1984472 | ||||||
| 24 h | 50 | 1954969 | 1955619 | 1182 | 0.0604 | ||
| 50 | 1954906 | ||||||
| 50 | 1956984 | ||||||
| 48 h | 50 | 1959292 | 1956961 | 2860 | 0.1461 | ||
| 50 | 1953769 | ||||||
| 50 | 1957822 | ||||||
| DAP | Initial | 100 | 1589486 | 1585358 | 3828 | 0.2414 | 0.0354 |
| 100 | 1581924 | ||||||
| 100 | 1584664 | ||||||
| 24 h | 100 | 1582857 | 1584432 | 2345 | 0.1480 | ||
| 100 | 1583312 | ||||||
| 100 | 1587128 | ||||||
| 48 h | 100 | 1586132 | 1585451 | 1889 | 0.1192 | ||
| 100 | 1586906 | ||||||
| 100 | 1583315 |
| Parameter | Observation | Penalty Points (Approx.) |
|---|---|---|
| Methanol (70%) in mobile phase | Hazardous organic solvent, but less problematic than acetonitrile | 12 |
| Phosphate buffer (30%) | Low environmental impact | 1 |
| Sample preparation using methanol | Additional solvent consumption | 4 |
| Energy consumption (HPLC instrument) | Conventional HPLC analysis | 1 |
| Occupational hazard | Standard laboratory handling | 0 |
| Waste generation (25 mL/analysis) | Between 10-100 mL | 5 |
| Total Penalty Points | 23 |
DISCUSSION
Box Behnken Design was employed for the optimisation of developed RP HPLC technique, which created 17 experimental runs with five repetitions of centre points, in order to evaluate effect, interactions of critical process parameters on dependent variables and to develop a model with sufficient statistical data. Composition of mobile phase solvents i. e. percentage of methanol, flow rate affects the retention time, no. of theoretical plates and peak area, separation of analyte etc. Optimising solvent ratio of mobile phase is very important for achieving good resolution, required retention time and optimum theoretical plates.42
The flow rate of diluent determines the time of analysis and peak shape of analytes. Maintaining appropriate flow rate can optimise the analysis time and resolution of peaks. pH is considered a key method attribute since it directly influences how analytes behave chemically, supports the robustness of the method, and helps maintain consistent and dependable analytical results. Therefore, careful regulation and monitoring of pH are crucial to ensure accuracy and proper method validation. Therefore, ratio of eluting solvent, flow rate, pH was identified as method performance characteristics. These method attributes were tested at three levels. BBD design was generated by Design Expert software. The optimised chromatographic conditions were selected by Derringer’s desirability (Figure 7).
Optimised mobile phase by Box Behnken design consisting of methanol and phosphate buffer having pH 2.8 in ratio 70:30 %v/v and flow rate was 0.8 mL/min. Cosmosil C18 column (250 mm x 4.6 mm; 5 µm) was utilised for chromatographic estimation. Detection wavelength was 227 nm which is isosbestic point. Sample volume 20 µL was used. Run time was 15 min. The different interaction and quadratic effects of the composition of mobile phase and flow rate on retention time, number of theoretical plates were analysed using ANOVA tool. ANOVA results of LIN by Box Behnken Design, response surface methodology for retention time had p-value 0.0001 and F statistical value 122.04. For DAP p-value was 0.0001 and F statistical value 237.71. For LIN theoretical plates, the developed model was statistically significant (p=0.0171; F-value = 5.55). The Adequate Precision value of 6.91 exceeded the minimum desirable value of 4, indicating an acceptable signal-to-noise ratio for navigation of the design space. However, compared with other responses, the model demonstrated moderate predictive strength, suggesting that additional experimental factors not included in the present design may contribute to variability in theoretical plate count. Therefore, the model should be interpreted as suitable for optimisation within the studied experimental domain, while caution should be exercised when extrapolating predictions beyond the investigated factor ranges. For theoretical plate of DAP, the p-value was 0.0001 and F statistical value was 62.32. The refined quadratic model for DAP theoretical plates exhibited excellent predictive capability, with an Adjusted R² of 0.9388 and a Predicted R² of 0.8840. The close agreement between these values (difference = 0.0548) indicates good model predictability and absence of significant overfitting. The Adequate Precision value of 27.03 demonstrates an adequate signal-to-noise ratio, confirming the suitability of the model for navigating the experimental design space and optimisation studies as shown in Table 2. These responses had p-value below 0.05 and F statistical value above 2.5 shows that the optimised RP HPLC method was significant. Adequate precision for all models was more than 4 (which is desirable), suggest adequate signal. The optimised procedure was evaluated and found to be in full compliance with ICH guidelines; all validation parameters met the required acceptance criteria. Linearity was confirmed over a selected concentration range for LIN and DAP, with coefficients of correlation (R2) were 0.9994 and 0.9996 for LIN and DAP correspondingly.
Compared with previously reported RP-HPLC methods for the simultaneous determination of LIN and DAP, the proposed AQbD-based method demonstrated several analytical advantages. Earlier methods generally relied on conventional trial-and-error optimisation approaches, whereas the present method employed a systematic AQbD strategy using Box-Behnken Design to establish a robust design space and identify method performance characteristics. The developed method exhibited excellent linearity within the selected concentration ranges, with correlation coefficients meets the acceptance criteria and sensitivity values (LOD and LOQ) comparable to those reported in previous studies. Precision studies showed %RSD values well below 2%, confirming excellent repeatability and intermediate precision. Accuracy was verified through recovery experiments, yielding recovery rates within acceptable limits. Precision studies were results in low RSD values, indicated that the method was reliable. Robustness was evaluated which reveals that there was no significant impact on the method performance. Furthermore, chromatographic performance parameters such as theoretical plate count, tailing factor, and resolution satisfied ICH acceptance criteria, indicating efficient separation and peak symmetry.
Greenness assessment was carried out by using AGREE, complex GAPI and analytical eco scale. Comparison with previously published RP-HPLC methods revealed that the proposed method provides analytical performance comparable to existing methods for simultaneous estimation of LIN and DAP. However, unlike the reported methods, the present work employed a Quality-by-Design (QbD) strategy using Box-Behnken Design for systematic method optimisation. Furthermore, the environmental sustainability of the method was comprehensively assessed using AGREE, Complex GAPI, and Analytical Eco-Scale tools. The Analytical Eco-Scale score of 77 and AGREE score of 0.61 indicate acceptable-to-excellent environmental performance. These features distinguish the present method from previously reported methods and support its suitability for routine quality control applications.
These findings suggest that the proposed AQbD-driven method provides a robust, reliable, and sustainable alternative to previously reported methods for routine quality control analysis of LIN and DAP in pharmaceutical dosage forms.
CONCLUSION
The reversed phase HPLC method was developed, optimised for concurrent quantification of LIN and DAP in their pure form and tablet dosage form by employing Box Behnken Design. The optimised technique was validated as per ICH guidelines. The analytical procedure validation results demonstrate that the developed RP-HPLC method is simple, precise, accurate, specific, and robust. Furthermore, the method exhibited acceptable environmental performance as demonstrated by AGREE, Complex GAPI, and Analytical Eco-Scale assessments. Therefore, the proposed method is suitable for routine quality control analysis of LIN and DAP in pharmaceutical formulations.
